Nanoparticle fabrication methods, systems, and materials for fabricating artificial red blood cells

ABSTRACT

A plurality of artificial red blood cell particles includes each particle of the plurality being substantially monodisperse and each particle having a largest common linear dimension of about 5 μm to about 10 μm. The particles can also have a modulus configured such that a particle of the plurality of particles can pass through a tube having an inner diameter of less than about 3 μm.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.12/374,182, filed Oct. 15, 2009, which is a National Phase ofInternational Application No. PCT/US2007/016935, filed Jul. 27, 2007,which claims priority to U.S. Provisional Application No. 60/833,736,filed Jul. 27, 2006, the entire contents of each are incorporated hereinby reference.

This application is also a continuation-in-part of PCT InternationalPatent Application Serial NO. PCT/US06/23722, filed Jun. 19, 2006, whichis incorporated herein by reference in its entirety.

INCORPORATION BY REFERENCE

All documents referenced herein are hereby incorporated by reference asif set forth in their entirety herein, as well as all references citedtherein.

GOVERNMENT INTEREST

This invention was made with U.S. Government support under Grant No.CHE-9876674 awarded by the National Science Foundation and from theOffice of Naval Research Grant No. N00014-02-1-0185. The government hascertain rights in the invention.

TECHNICAL FIELD

Generally, this invention relates to micro and/or nano scale particlefabrication. More particularly, the micro and/or nano scale particlesare fabricated to mimic red blood cells.

ABBREVIATIONS

° C.=degrees Celsius

cm=centimeter.

DBTDA=dibutyltin diacetate

DMA=dimethylacrylate

DMPA=2,2-dimethoxy-2-phenylacetophenone

EIM=2-isocyanatoethyl methacrylate

FEP=fluorinated ethylene propylene

Freon 113=1,1,2-trichlorotrifluoroethane

g=grams

h=hours

Hz=hertz

IL=imprint lithography

kg=kilograms

kHz=kilohertz

kPa=kilopascal

MCP=microcontact printing

MEMS=micro-electro-mechanical system

MHz=megahertz

MIMIC=micro-molding in capillaries

mL=milliliters

mm=millimeters

mmol=millimoles

mN=milli-Newton

m.p.=melting point

mW=milliwatts

NCM=nano-contact molding

NIL=nanoimprint lithography

nm=nanometers

PDMS=polydimethylsiloxane

PEG=poly(ethylene glycol)

PFPE=perfluoropolyether

PLA=poly(lactic acid)

PP=polypropylene

Ppy=poly(pyrrole)

psi=pounds per square inch

PVDF=poly(vinylidene fluoride)

PTFE=polytetrafluoroethylene

SAMIM=solvent-assisted micro-molding

SEM=scanning electron microscopy

S-FIL=“step and flash” imprint lithography

Si=silicon

Tg=glass transition temperature

Tm=crystalline melting temperature

TMPTA=trimethylolpropane triacrylate

μm=micrometers

UV=ultraviolet

W=watts

BACKGROUND

Mammalian red blood cells are critical for the delivery of oxygen tobody tissues and the exchange of carbon dioxide from body tissues. Onecritical feature of red blood cells (RBC) is their ability to severelydeform in shape to pass through intercellular gaps of sinusoids in thespleen and capillaries. Disorders of red blood cells can enhancerigidification of red blood cells and reduce their ability to passthrough intercellular gaps and capillaries. Such rigidification is a keyfeature of the biology and pathophysiology of malaria (Miller, et al.Nature (2002); Cooke, et al. Adv. Parasitology (2001); and Glenister,et. al Blood (2002); each of which is incorporated herein by reference).Sickle cell anemia is another RBC-based condition which is caused byelongated RBCs. Furthermore, over time RBCs stiffen and aged red bloodcells are removed from the body after about 120 days. Therefore, thereexists a need to fabricate an artificial RBC which can deform in size topass through intercellular gaps and capillaries and carry and exchangeoxygen with carbon dioxide.

SUMMARY

According to some embodiments, an artificial red blood cell includes aplurality of particles where each particle of the plurality of particlesis substantially monodisperse. In some embodiments, each particle has alargest linear dimension of about 5 μm to about 10 μm and a modulus lessthan about 1 MPa. In some embodiments, a particle of the plurality ofparticles can pass through a tube having an inner diameter of less thanabout 3 μm.

According to some embodiments, an artificial red blood cell includes aplurality of substantially monodisperse particles, where each particlehas substantially a disc shape. In some embodiments, each particle has adiameter of about 5 μm to about 10 μm. In some embodiments, eachparticle has a porosity configured to give the particle a modulus suchthat the particle can pass through a tube having an inner diameter ofless than about 3 μm.

According to some embodiments, the particles include poly(ethyleneglycol). In some embodiments, the particles include perfluoropolyether.

According to some embodiments, the particles may include surfacefunctionality and/or cargo. In some embodiments, the cargo is capable ofbinding and releasing oxygen.

In some embodiments, particles are used to obtain sustained andmodulated drug delivery. The design and development of such systems,mathematical modeling of transport from these systems and the in vivouse of these devices accumulate to their impact and potential use in avariety of disease states. Measurement of drug distribution in vasculartissue using quantitative fluorescence microscopy of the particlesdisclosed herein is one such system.

In some embodiments, particles can be fabricated from biocompatiblematerials with solubility and/or philicity control. The mesh density andcharge can be altered to control factors such as modulus and release ofcargo. In some embodiments, particles can be designed with stimulateddegradation for cargo release. Due to the unique manufacturing method,described in pending PCT application PCT/US06/23722 filed on Jun. 19,2006 which is incorporated herein by reference in its entirety includingall references cited therein, monodisperse particles can be made withshape and size specificity.

According to some embodiments, particles may carry a wide variety ofcargos. Particles may incorporate therapeutics, such as small molecules,proteins, oligos, siRNAs, and pDNA, imaging beacons for PET, SPECT, MR,and ultrasound, as well as organelles. In some embodiments, no chemicalmodification of the cargo is needed. High loadability is also possible.

In some embodiments, particles are amenable to surface functionalizationfor targeting and enhanced circulation. In some embodiments, ligands onthe surface allow for tailored bioavailability and enhancedelectrostatic or steric stabilization. Non-spherical particles have aunique ability to increase the number of surface ligands per cargovolume. The particles are also amenable to all dosage forms, includinginjectables, oral, inhalation, and dermatological.

BRIEF DESCRIPTION OF THE DRAWINGS

Reference is made to the accompanying drawings in which are shownillustrative embodiments of the presently disclosed subject matter, fromwhich its novel features and advantages will be apparent.

FIGS. 1A-1D are a schematic representation of an embodiment of thepresently disclosed method for preparing a patterned template.

FIGS. 2A-2F are a schematic representation of the presently disclosedmethod for forming one or more micro- and/or nanoscale particles.

FIGS. 3A-3F are schematic representations of one embodiment of a methodof the presently disclosed subject matter for harvesting particles froman article.

FIGS. 4A-4G are schematic representations of one embodiment of a methodof the presently disclosed subject matter for harvesting particles froman article.

FIGS. 5A-5C are schematic representations of red blood cells. FIG. 5Arepresents an SEM image of red blood cells in a capillary. FIG. 5Brepresents a schematic diagram of a red blood cell. FIG. 5C representsan image of red blood cells in a 7 μm capillary.

FIG. 6 is a schematic representation of a substantially disc-shapedparticle with cargo.

FIG. 7 is a schematic representation of various cargo andfunctionalization which may be applied to a particle.

FIG. 8 is a schematic representation of the use of particles for immuneresponse.

FIG. 9 is a schematic addressing use of particles to deliver organelles.

FIGS. 10A and 10B are an atomic force micrograph of mold fabricationfrom brush polymer masters. FIG. 10A is a brush polymer master. FIG. 10Bis a PFPE-DMA mold templated from a brush polymer master.

FIGS. 11A-11D are schematic representations of one embodiment of amethod for functionalizing particles of the presently disclosed subjectmatter.

FIGS. 12A-12D are schematic representations of one embodiment of oneprocess of the presently disclosed subject matter for harvestingparticles from an article.

FIG. 13 is a scanning electron micrograph of a silicon master including200 nm trapezoidal patterns.

FIG. 14 is a scanning electron micrograph of 200-nm isolated trapezoidalparticles of poly(ethylene glycol) (PEG) diacrylate.

FIG. 15 is a scanning electron micrograph of a silicon master including500 nm conical patterns that are <50 nm at the tip.

FIG. 16 is a scanning electron micrograph of 500-nm isolated conicalparticles of PEG diacrylate.

FIG. 17 is a scanning electron micrograph of a silicon master including3-μm arrow-shaped patterns.

FIG. 18 is a scanning electron micrograph of 3-μm isolated arrow-shapedparticles of PEG diacrylate.

FIG. 19 is a scanning electron micrograph of 200-nm×750-nm×250-nmrectangular shaped particles of PEG diacrylate.

FIG. 20 is a scanning electron micrograph of 200-nm isolated trapezoidalparticles of trimethylolpropane triacrylate (TMPTA).

FIG. 21 is a scanning electron micrograph of 500-nm isolated conicalparticles of TMPTA.

FIG. 22 is a scanning electron micrograph of 500-nm isolated conicalparticles of TMPTA, which have been printed using an embodiment of thepresently described non-wetting imprint lithography method and harvestedmechanically using a doctor blade.

FIG. 23 is a scanning electron micrograph of 200-nm isolated trapezoidalparticles of poly(lactic acid) (PLA).

FIG. 24 is a scanning electron micrograph of 200-nm isolated trapezoidalparticles of poly(lactic acid) (PLA), which have been printed using anembodiment of the presently described non-wetting imprint lithographymethod and harvested mechanically using a doctor blade.

FIG. 25 is a scanning electron micrograph of 3-μm isolated arrow-shapedparticles of PLA.

FIG. 26 is a scanning electron micrograph of 500-nm isolatedconical-shaped particles of PLA.

FIG. 27 is a scanning electron micrograph of 200-nm isolated trapezoidalparticles of poly(pyrrole) (Ppy).

FIG. 28 is a scanning electron micrograph of 3-μm arrow-shaped Ppyparticles.

FIG. 29 is a scanning electron micrograph of 500-nm conical shaped Ppyparticles.

FIGS. 30A-30C are fluorescence confocal micrographs of 200-nm isolatedtrapezoidal particles of PEG diacrylate that contain fluorescentlytagged DNA. FIG. 30A is a fluorescent confocal micrograph of 200 nmtrapezoidal PEG nanoparticles which contain 24-mer DNA strands that aretagged with CY-3. FIG. 30B is optical micrograph of the 200-nm isolatedtrapezoidal particles of PEG diacrylate that contain fluorescentlytagged DNA. FIG. 30C is the overlay of the images provided in FIGS. 30Aand 30B, showing that every particle contains DNA.

FIG. 31 is a scanning electron micrograph of fabrication of 200-nmPEG-diacrylate nanoparticles using “double stamping.”

FIG. 32 shows doxorubicin containing particles after removal from atemplate according to an embodiment of the presently disclosed subjectmatter.

FIG. 33 shows a structure patterned with nano-cylindrical shapesaccording to an embodiment of the presently disclosed subject matter.

FIGS. 34A and 34B show cube-shaped PEG particles fabricated by a dippingmethod according to an embodiment of the present invention.

FIG. 35 is an atomic force micrograph image of 140-nm lines of TMPTAseparated by distance of 70 nm that were fabricated using a PFPE mold.

FIGS. 36A and 36B are a scanning electron micrograph of mold fabricationfrom electron-beam lithographically generated masters. FIG. 36A is ascanning electron micrograph of silicon/silicon oxide masters of 3micron arrows. FIG. 36B is a scanning electron micrograph ofsilicon/silicon oxide masters of 200-nm×800-nm bars.

FIGS. 37A and 37B are an optical micrographic Image of mold fabricationfrom photoresist masters. FIG. 37A is a SU-8 master. FIG. 37B is aPFPE-DMA mold templated from a photolithographic master.

FIGS. 38A and 38B are an atomic force micrograph of mold fabricationfrom Tobacco Mosaic Virus templates. FIG. 38A is a master. FIG. 38B is aPFPE-DMA mold templated from a virus master.

FIGS. 39A and 39B are an atomic force micrograph of mold fabricationfrom block copolymer micelle masters. FIG. 39A is apolystyrene-polyisoprene block copolymer micelle. FIG. 39B is a PFPE-DMAmold templated from a micelle master.

FIG. 40 shows an SEM micrograph of 2×2×1 μm positively charged DEDSMAparticles according to an embodiment of the present invention.

FIG. 41 shows a fluorescent micrograph of 2×2×1 μm positively chargedDEDSMA particles according to an embodiment of the present invention.

FIG. 42 shows a fluorescence micrograph of calcein cargo incorporatedinto 2 μm DEDSMA particles according to an embodiment of the presentinvention.

FIG. 43 shows 2×2×1 μm pDNA containing positively charged DEDSMAparticles: Top Left: SEM, Top Right: DIC, Bottom Left: Particle-boundPolyfluor 570 fluorescence, Bottom Right: Fluorescein-labelled controlplasmid fluorescence according to an embodiment of the presentinvention.

FIG. 44 shows 2×2×1 μm pDNA containing positively charged PEG particles:Top Left: SEM, Top Right: DIC, Bottom Left: Particle-bound Polyfluor 570fluorescence, Bottom Right: Fluorescein-labelled control plasmidfluorescence according to an embodiment of the present invention.

FIG. 45 shows master templates containing 200 nm cylindrical shapes withvarying aspect ratios according to an embodiment of the presentinvention.

FIG. 46 shows a scanning electron micrograph (at a 45° angle) ofharvested neutral PEG-composite 200 nm (aspect ratio=1:1) particles onthe poly(cyanoacrylate) harvesting layer according to an embodiment ofthe present invention.

FIG. 47 shows confocal micrographs of cellular uptake of purified PRINTPEG-composite particles into NIH 3T3 cells—trends in amount of cationiccharge according to an embodiment of the present invention.

FIG. 48 shows toxicity results obtained from an MT assay on varying boththe amount of cationic charge incorporated into a particle matrix, aswell as an effect of particle concentration on cellular uptake accordingto an embodiment of the present invention.

FIG. 49 shows confocal micrographs of cellular uptake of PRINT PEGparticles into NIH 3T3 cells while the inserts show harvested particleson medical adhesive layers prior to cellular treatment according to anembodiment of the present invention.

FIG. 50 shows a reaction scheme for conjugation of a radioactivelylabeled moiety to PRINT particles according to an embodiment of thepresent invention.

FIG. 51 shows fabrication of pendant gadolinium PEG particles accordingto an embodiment of the present invention.

FIG. 52 shows formation of a particle containing CDI linker according toan embodiment of the present invention.

FIG. 53 shows tethering avidin to a CDI linker according to anembodiment of the present invention.

FIG. 54 shows fabrication of PEG particles that target an HER2 receptoraccording to an embodiment of the present invention.

FIG. 55 shows fabrication of PEG particles that target non-Hodgkin'slymphoma according to an embodiment of the present invention.

FIG. 56 shows a method of dipping a patterned template to introduce asubstance into recesses of the patterned template according to anembodiment of the present invention.

FIG. 57 shows particles formed from methods described herein andreleased from a mold according to an embodiment of the presentinvention.

FIG. 58 shows a method of flowing a substance across a patternedtemplate surface to introduce the substance into recesses of thepatterned template according to an embodiment of the present invention.

FIG. 59 shows further particles formed from methods described herein andreleased from a mold according to an embodiment of the presentinvention.

DETAILED DESCRIPTION

The presently disclosed subject matter will now be described more fullyhereinafter with reference to the accompanying Examples, in whichrepresentative embodiments are shown. The presently disclosed subjectmatter can, however, be embodied in different forms and should not beconstrued as limited to the embodiments set forth herein. Rather, theseembodiments are provided so that this disclosure will be thorough andcomplete, and will fully convey the scope of the embodiments to thoseskilled in the art.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this presently described subject matter belongs. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety.

Throughout the specification and claims, a given chemical formula orname shall encompass all optical and stereoisomers, as well as racemicmixtures where such isomers and mixtures exist.

I. Formation of Isolated Micro- and/or Nanoparticles

In some embodiments, the presently disclosed subject matter providesisolated micro- and/or nanoparticles and methods for making the isolatedmicro- and/or nanoparticles. In some embodiments, the process for makingthe isolated micro and/or nanoparticles includes initially forming apatterned substrate. Turning now to FIG. 1A, a patterned master 100 isprovided. Patterned master 100 includes a plurality of non-recessedsurface areas 102 and a plurality of recesses 104. In some embodiments,patterned master 100 includes an etched substrate, such as a siliconwafer, which is etched in the desired pattern to form patterned master100.

Referring now to FIG. 1B, a liquid material 106, for example, a liquidfluoropolymer composition, such as a PFPE-based precursor, is thenpoured onto patterned master 100. Liquid material 106 is treated bytreating process T_(r), for example exposure to UV light, actinicradiation, or the like, thereby forming a treated liquid material 108 inthe desired pattern.

Referring now to FIGS. 1C and 1D, a force Fr is applied to treatedliquid material 108 to remove it from patterned master 100. As shown inFIGS. 1C and 1D, treated liquid material 108 includes a plurality ofrecesses 110, which are mirror images of the plurality of non-recessedsurface areas 102 of patterned master 100. Continuing with FIGS. 1C and1D, treated liquid material 108 includes a plurality of first patternedsurface areas 112, which are mirror images of the plurality of recesses104 of patterned master 100. Treated liquid material 108 can now be usedas a patterned template for soft lithography and imprint lithographyapplications. Accordingly, treated liquid material 108 can be used as apatterned template for the formation of isolated micro- andnanoparticles.

In some embodiments, the patterned template includes a patternedtemplate formed by a replica molding process. In some embodiments, thereplica molding process includes: providing a master template;contacting a liquid material with the master template; and curing theliquid material to form a patterned template.

In some embodiments, the master template includes, without limitation,one or more of a template formed from a lithography process, a naturallyoccurring template, combinations thereof, or the like. In someembodiments, the natural template is selected from one of a biologicalstructure and a self-assembled structure. In some embodiments, the oneof a biological structure and a self-assembled structure is selectedfrom the group including a naturally occurring crystal, an enzyme, avirus, a protein, a micelle, and a tissue surface.

In some embodiments, the method includes modifying the patternedtemplate surface by a surface modification step. In some embodiments,the surface modification step is selected from the group including aplasma treatment, a chemical treatment, and an adsorption process. Insome embodiments, the adsorption process includes adsorbing moleculesselected from the group including a polyelectrolyte, apoly(vinylalcohol), an alkylhalosilane, and a ligand.

Referring now to FIG. 2A, in some embodiments, a substrate 200, forexample, a silicon wafer, is treated or is coated with a non-wettingmaterial 202. In some embodiments, non-wetting material 202 includes anelastomer (such a solvent resistant elastomer, including but not limitedto a PFPE elastomer) that can be further exposed to UV light and curedto form a thin, non-wetting layer on the surface of substrate 200.Substrate 200 also can be made non-wetting by treating substrate 200with non-wetting agent 202, for example a small molecule, such as analkyl- or fluoroalkyl-silane, or other surface treatment. Continuingwith FIG. 2A, a droplet 204 of a curable resin, a monomer, or a solutionfrom which the desired particles will be formed is then placed on thecoated substrate 200.

Referring now to FIG. 2A and FIG. 2B, patterned template 108 (as shownin FIG. 1D) is then contacted with droplet 204 of a particle precursormaterial so that droplet 204 fills the plurality of recessed areas 110of patterned template 108.

Referring now to FIGS. 2C and 2D, in some embodiments a force F_(a) isapplied to patterned template 108. While not wishing to be bound by anyparticular theory, once force F_(a) is applied, the affinity ofpatterned template 108 for non-wetting coating or surface treatment 202on substrate 200 in combination with the non-wetting behavior ofpatterned template 108 and surface treated or coated substrate 200causes droplet 204 to be excluded from all areas except for recessedareas 110. Further, in embodiments essentially free of non-wetting orlow wetting material 202 with which to sandwich droplet 204, a “scum”layer forms that interconnects the objects being stamped.

Continuing with FIGS. 2C and 2D, the particle precursor material fillingrecessed areas 110, e.g., a resin, monomer, solvent, combinationsthereof, or the like, is then treated by a treating process T_(r), e.g.,photocured, UV-light treated, or actinic radiation treated, throughpatterned template 108 or thermally cured while under pressure, to forma plurality of micro- and/or nanoparticles 206. In some embodiments, amaterial, including but not limited to a polymer, an organic compound,or an inorganic compound, can be dissolved in a solvent, patterned usingpatterned template 108, and the solvent can be released.

Continuing with FIGS. 2C and 2D, once the material filling recessedareas 110 is treated, patterned template 108 is removed from substrate200. Micro- and/or nanoparticles 206 are confined to recessed areas 110of patterned template 108. In some embodiments, micro- and/ornanoparticles 206 can be retained on substrate 200 in defined regionsonce patterned template 108 is removed.

According to other embodiments, substrate 200 is not utilized and thematerial filling recessed areas 110 that becomes micro and/ornanoparticles 206 enters recessed areas 110 through capillary force,wetting characteristics, passive filling, active filling, or the like asdescribed elsewhere herein.

Referring now to FIGS. 2D and 2E, micro- and/or nanoparticles 206 can beremoved from patterned template 108 to provide freestanding particles bya variety of methods, which include but are not limited to: (1) applyingpatterned template 108 to a surface that has an affinity for theparticles 206; (2) deforming patterned template 108, or using othermechanical methods, including sonication, in such a manner that theparticles 206 are naturally released from patterned template 108; (3)swelling patterned template 108 reversibly with supercritical carbondioxide or another solvent that will extrude the particles 206; (4)washing patterned template 108 with a solvent that has an affinity forthe particles 206 and will wash them out of patterned template 108; (5)applying patterned template 108 to a liquid that when hardenedphysically entraps particles 206; (6) applying patterned template 108 toa material that when hardened has a chemical and/or physical interactionwith particles 206.

In some embodiments, the method of producing and harvesting particlesincludes a batch process. In some embodiments, the batch process isselected from one of a semi-batch process and a continuous batchprocess. Referring now to FIG. 2F, an embodiment of the presentlydisclosed subject matter wherein particles 206 are produced in acontinuous process is schematically presented. An apparatus 199 isprovided for carrying out the process. Indeed, while FIG. 2Fschematically presents a continuous process for particles, apparatus 199can be adapted for batch processes, and for providing a pattern on asubstrate continuously or in batch, in accordance with the presentlydisclosed subject matter and based on a review of the presentlydisclosed subject matter by one of ordinary skill in the art.

Continuing, then, with FIG. 2F, droplet 204 of liquid material isapplied to substrate 200′ via reservoir 203. Substrate 200′ can becoated or not coated with a non-wetting agent. Substrate 200′ andpattern template 108′ are placed in a spaced relationship with respectto each other and are also operably disposed with respect to each otherto provide for the conveyance of droplet 204 between patterned template108′ and substrate 200′. Conveyance is facilitated through the provisionof pulleys 208, which are in operative communication with controller201. By way of representative non-limiting examples, controller 201 caninclude a computing system, appropriate software, a power source, aradiation source, and/or other suitable devices for controlling thefunctions of apparatus 199. Thus, controller 201 provides for power forand other control of the operation of pulleys 208 to provide for theconveyance of droplet 204 between patterned template 108′ and substrate200′. Particles 206 are formed and treated between substrate 200′ andpatterned template 108′ by a treating process T_(R), which is alsocontrolled by controller 201. Particles 206 are collected in aninspecting device 210, which is also controlled by controller 201.Inspecting device 210 provides for one of inspecting, measuring, andboth inspecting and measuring one or more characteristics of particles206. Representative examples of inspecting devices 210 are disclosedelsewhere herein.

By way of further exemplifying embodiments of particle harvestingmethods described herein, reference is made to FIGS. 3A-3F and FIGS.4A-4G. In FIGS. 3A-3C and FIGS. 4A-4C particles which are produced inaccordance with embodiments described herein remain in contact with anarticle 3700, 3800. The article 3700, 3800 can have an affinity forparticles 3705 and 3805, respectively, or the particles can simpleremain in the mold recesses following fabrication of the particlestherein. In one embodiment, article 3700 is a patterned template or moldas described herein and article 3800 is a substrate as described herein.

Referring now to FIGS. 3D-3F and FIGS. 4D-4G, material 3720, 3820 havingan affinity for particles 3705, 3805 is put into contact with particles3705, 3805 while particles 3705, 3805 remain in communication witharticles 3700, 3800. In the embodiment of FIG. 3D, material 3720 isdisposed on surface 3710. In the embodiment of FIG. 4D, material 3820 isapplied directly to article 3800 having particles 3820. As illustratedin FIGS. 3E, 4D in some embodiments, article 3700, 3800 is put inengaging contact with material 3720, 3820. In one embodiment material3720, 3820 is thereby dispersed to coat at least a portion ofsubstantially all of particles 3705, 3805 while particles 3705, 3805 arein communication with article 3700, 3800 (e.g., a patterned template).In one embodiment, illustrated in FIGS. 3F and 4F, articles 3700, 3800are substantially disassociated with material 3720, 3820. In oneembodiment, material 3720, 3820 has a higher affinity for particles3705, 3805 than any affinity between article 3700, 3800 and particles3705, 3805. In FIGS. 3F and 4F, the disassociation of article 3700, 3800from material 3720, 3820 thereby releases particles 3705, 3805 fromarticle 3700, 3800 leaving particles 3705, 3805 associated with material3720, 3820.

In one embodiment material 3720, 3820 has an affinity for particles 3705and 3805. For example, material 3720, 3820 can include an adhesive orsticky surface such that when it is applied to particles 3705 and 3805the particles remain associated with material 3720, 3820 rather thanwith article 3700, 3800. In other embodiments, material 3720, 3820undergoes a transformation after it is brought into contact with article3700, 3800. In some embodiments that transformation is an inherentcharacteristic of material 3705, 3805. In other embodiments, material3705, 3805 is treated to induce the transformation. For example, in oneembodiment material 3720, 3820 is an epoxy that hardens after it isbrought into contact with article 3700, 3800. Thus, when article 3700,3800 is pealed away from the hardened epoxy, particles 3705, 3805 remainengaged with the epoxy and not article 3700, 3800. In other embodiments,material 3720, 3820 is water that is cooled to form ice. Thus, whenarticle 3700, 3800 is stripped from the ice, particles 3705, 3805 remainin communication with the ice and not article 3700, 3800. In oneembodiment, the particle in connection with ice can be melted to createa liquid with a concentration of particles 3705, 3805. In someembodiments, material 3705, 3805 include, without limitation, one ormore of a carbohydrate, an epoxy, a wax, polyvinyl alcohol, polyvinylpyrrolidone, polybutyl acrylate, a polycyano acrylate and polymethylmethacrylate. In some embodiments, material 3720, 3820 includes, withoutlimitation, one or more of liquids, solutions, powders, granulatedmaterials, semi-solid materials, suspensions, combinations thereof, orthe like.

In some embodiments, the plurality of recessed areas includes aplurality of cavities. In some embodiments, the plurality of cavitiesincludes a plurality of structural features. In some embodiments, theplurality of structural features have a dimension ranging from about 10microns to about 1 nanometer in size. In some embodiments, the pluralityof structural features have a dimension ranging from about 1 micron toabout 100 nm in size. In some embodiments, the plurality of structuralfeatures have a dimension ranging from about 100 nm to about 1 mm insize. In some embodiments, the plurality of structural features have adimension in both the horizontal and vertical plane.

In some embodiments, the method of producing particles includespositioning the patterned template and the substrate in a spacedrelationship to each other such that the patterned template surface andthe substrate face each other in a predetermined alignment.

In some embodiments, an article is contacted with the layer of liquidmaterial and a force is applied to the article to thereby remove theliquid material from the one of the patterned material and thesubstrate. In some embodiments, the article is selected from the groupincluding a roller, a “squeegee” blade type device, a nonplanarpolymeric pad, combinations thereof, or the like. In some embodiments,the liquid material is removed by some other mechanical apparatus.

In some embodiments, the contacting of the patterned template surfacewith the substrate forces essentially all of the disposed liquidmaterial from between the patterned template surface and the substrate.

In some embodiments, the treating of the liquid material includes aprocess selected from the group including a thermal process, a phasechange, an evaporative process, a photochemical process, and a chemicalprocess.

In some embodiments, the mechanical force is applied by contacting oneof a doctor blade and a brush with the one or more particles. In someembodiments, the mechanical force is applied by ultrasonics, megasonics,electrostatics, or magnetics means.

In some embodiments, the method includes harvesting or collecting theparticles. In some embodiments, the harvesting or collecting of theparticles includes a process selected from the group including scrapingwith a doctor blade, a brushing process, a dissolution process, anultrasound process, a megasonics process, an electrostatic process, anda magnetic process. In some embodiments, the harvesting or collecting ofthe particles includes applying a material to at least a portion of asurface of the particle wherein the material has an affinity for theparticles. In some embodiments, the material includes an adhesive orsticky surface. In some embodiments, the material includes, withoutlimitation, one or more of a carbohydrate, an epoxy, a wax, polyvinylalcohol, polyvinyl pyrrolidone, polybutyl acrylate, a polycyanoacrylate, a polyhydroxyethyl methacrylate, a polyacrylic acid andpolymethyl methacrylate. In some embodiments, the harvesting orcollecting of the particles includes cooling water to form ice (e.g., incontact with the particles). In some embodiments, the presentlydisclosed subject matter describes a particle or plurality of particlesformed by the methods described herein. In some embodiments, theplurality of particles includes a plurality of monodisperse particles.According to some embodiments, monodisperse particles are particles thathave a physical characteristic that falls within a normalized sizedistribution tolerance limit. According to some embodiments, the sizecharacteristic, or paramater, that is analyzed is the surface area,circumference, a linear dimension, mass, volume, three dimensionalshape, shape, or the like.

According to some embodiments, the particles have a normalized sizedistribution of between about 0.80 and about 1.20, between about 0.90and about 1.10, between about 0.95 and about 1.05, between about 0.99and about 1.01, between about 0.999 and about 1.001, combinationsthereof, and the like. Furthermore, in other embodiments the particleshave a mono-dispersity. According to some embodiments, dispersity iscalculated by averaging a dimension of the particles. In someembodiments, the dispersity is based on, for example, surface area,length, width, height, mass, volume, porosity, combinations thereof, andthe like.

In some embodiments, the particle or plurality of particles is selectedfrom the group including a semiconductor device, a crystal, a drugdelivery vector, a gene delivery vector, a disease detecting device, adisease locating device, a photovoltaic device, a porogen, a cosmetic,an electret, an additive, a catalyst, a sensor, a detoxifying agent, anabrasive, such as a CMP, a micro-electro-mechanical system (MEMS), acellular scaffold, a taggant, a pharmaceutical agent, and a biomarker.In some embodiments, the particle or plurality of particles include afreestanding structure.

According to some embodiments, a material can be incorporated into aparticle composition or a particle according to the present invention,to treat or diagnose diseases including, but not limited to, Allergies;Anemia; Anxiety Disorders; Autoimmune Diseases; Back and Neck Injuries;Birth Defects; Blood Disorders; Bone Diseases; Cancers; CirculationDiseases; Dental Conditions; Depressive Disorders; Digestion andNutrition Disorders; Dissociative Disorders; Ear Conditions; EatingDisorders; Eye Conditions; Foodborne Illnesses; GastrointestinalDiseases; Genetic Disorders; Heart Diseases; Heat and Sun RelatedConditions; Hormonal Disorders; Impulse Control Disorders; InfectiousDiseases; Insect Bites and Stings; Institutes; Kidney Diseases;Leukodystrophies; Liver Diseases; Mental Health Disorders; MetabolicDiseases; Mood Disorders; Neurological Disorders; Organizations;Personality Disorders; Phobias; Pregnancy Complications; Prion Diseases;Prostate Diseases; Registries; Respiratory Diseases; Sexual Disorders;Sexually Transmitted Diseases; Skin Conditions; Sleep Disorders;Speech-Language Disorders; Sports Injuries; Thyroid Diseases; TropicalDiseases; Vestibular Disorders; Waterborne Illnesses; and other diseasessuch as found at: http://www.mic.ki.se/Diseases/Alphalist.html, which isincorporated herein by reference in its entirety including eachreference cited therein.

In some embodiments, the method of producing particles includestailoring the chemical composition of these materials and controllingthe reaction conditions, whereby it is then possible to organize thebiorecognition motifs so that the efficacy of the particle is optimized.In some embodiments, the particles are designed and synthesized so thatrecognition elements are located on the surface of the particle in sucha way to be accessible to cellular binding sites, wherein the core ofthe particle is preserved to contain bioactive agents, such astherapeutic molecules. In some embodiments, a non-wetting imprintlithography method is used to fabricate the objects, wherein the objectsare optimized for a particular application by incorporating functionalmotifs, such as biorecognition agents, into the object composition. Insome embodiments, the method further includes controlling the microscaleand nanoscale structure of the object by using methods selected from thegroup including self-assembly, stepwise fabrication procedures, reactionconditions, chemical composition, crosslinking, branching, hydrogenbonding, ionic interactions, covalent interactions, and the like. Insome embodiments, the method further includes controlling the microscaleand nanoscale structure of the object by incorporating chemicallyorganized precursors into the object. In some embodiments, thechemically organized precursors are selected from the group includingblock copolymers and core-shell structures.

IA. Micro and Nano Particles

Dimensions

According to some embodiments of the presently disclosed subject matter,a particle is formed that has a shape corresponding to a mold (e.g., theparticle has a shape reflecting the shape of the mold within which theparticle was formed) having a desired shape and is less than about 100μm in a given dimension (e.g. minimum, intermediate, or maximumdimension). In some embodiments, the particle is a nano-scale particle.According to some embodiments, the nano-scale particle has a dimension,such as a diameter or linear measurement that is less than 500 micron.The dimension can be measured across the largest portion of the particlethat corresponds to the parameter being measured. In other embodiments,the dimension is less than 250 micron. In other embodiments, thedimension is less than 100 micron. In other embodiments, the dimensionis less than 50 micron. In other embodiments, the dimension is less than10 micron. In other embodiments, the dimension is between 1 nm and 1,000nm. In some embodiments, the dimension is less than 1,000 nm. In otherembodiments, the dimension is between 1 nm and 500 nm. In yet otherembodiments, the dimension is between 1 nm and 100 nm.

According to some embodiments, particles formed in the patternedtemplates described herein are less than about 10 μm in a dimension. Inother embodiments, the particle is between about 10 μm and about 1 μm indimension. In yet further embodiments, the particle is less than about 1μm in dimension. According to some embodiments the particle is betweenabout 1 nm and about 500 nm in a dimension. According to otherembodiments, the particle is between about 10 nm and about 200 nm in adimension. In still further embodiments, the particle is between about80 nm and 120 nm in a dimension. According to still more embodiments theparticle is between about 20 nm and about 120 nm in dimension. Thedimension of the particle can be a predetermined dimension, across-sectional diameter, a circumferential dimension, or the like.

According to further embodiments, the particles include patternedfeatures that are about 2 nm in a dimension. In still furtherembodiments, the patterned features are between about 2 nm and about 200nm. In other embodiments, the particle is less than about 80 nm in awidest dimension.

According to other embodiments, the particles produced by the methodsand materials of the presently disclosed subject matter have a polydispersion index (i.e., normalized size distribution) of between about0.80 and about 1.20, between about 0.90 and about 1.10, between about0.95 and about 1.05, between about 0.99 and about 1.01, between about0.999 and about 1.001, combinations thereof, and the like. Furthermore,in other embodiments the particle has a mono-dispersity. According tosome embodiments, dispersity is calculated by averaging a dimension ofthe particles. In some embodiments, the dispersity is based on, forexample, surface area, length, width, height, mass, volume, porosity,combinations thereof, and the like.

According to other embodiments, particles of many predetermined regularand irregular shape and size configurations can be made with thematerials and methods of the presently disclosed subject matter.Examples of representative particle shapes that can be made using thematerials and methods of the presently disclosed subject matter include,but are not limited to, non-spherical, spherical, viral shaped, bacteriashaped, cell shaped, rod shaped (e.g., where the rod is less than about200 nm in diameter), chiral shaped, right triangle shaped, fiat shaped(e.g., with a thickness of about 2 nm, disc shaped with a thickness ofgreater than about 2 nm, or the like), bi-concave disc shaped, annularshaped with or without an opening in its center, boomerang shaped,combinations thereof, and the like.

Composition

The particle can be of an organic material or an inorganic material andcan be one uniform compound or component or a mixture of compounds orcomponents. In some embodiments, an organic material molded with thematerials and methods of the present invention includes a material thatincludes a carbon molecule. According to some embodiments, the particlecan be of a high molecular weight material. According to someembodiments, a particle is composed of a matrix that has a predeterminedsurface energy. In some embodiments, the material that forms theparticle includes more than about 50 percent liquid. In someembodiments, the material that forms the particle includes less thanabout 50 percent liquid. In some embodiments, the material that formsthe particle includes less than about 10 percent liquid.

In some embodiments, the material from which the particles are formedincludes, without limitation, one or more of a polymer, a liquidpolymer, a solution, a monomer, a plurality of monomers, apolymerization initiator, a polymerization catalyst, an inorganicprecursor, an organic material, a natural product, a metal precursor, apharmaceutical agent, a tag, a magnetic material, a paramagneticmaterial, a ligand, a cell penetrating peptide, a porogen, a surfactant,a plurality of immiscible liquids, a solvent, a charged species,combinations thereof, or the like.

In some embodiments, the monomer includes butadienes, styrenes, propene,acrylates, methacrylates, vinyl ketones, vinyl esters, vinyl acetates,vinyl chlorides, vinyl fluorides, vinyl ethers, acrylonitrile,methacrylonitrile, acrylamide, methacrylamide allyl acetates, fumarates,maleates, ethylenes, propylenes, tetrafluoroethylene, ethers,isobutylene, fumaronitrile, vinyl alcohols, acrylic acids, amides,carbohydrates, esters, urethanes, siloxanes, formaldehyde, phenol, urea,melamine, isoprene, isocyanates, epoxides, bisphenol A, alcohols,chlorosilanes, dihalides, dienes, alkyl olefins, ketones, aldehydes,vinylidene chloride, anhydrides, saccharide, acetylenes, naphthalenes,pyridines, tactams, lactones, acetals, thiiranes, episulfide, peptides,derivatives thereof, and combinations thereof.

In yet other embodiments, the polymer includes polyamides, proteins,polyesters, polystyrene, polyethers, polyketones, polysulfones,polyurethanes, polysiloxanes, polysilanes, cellulose, amylose,polyacetals, polyethylene, glycols, poly(acrylate)s,poly(methacrylate)s, poly(vinyl alcohol), poly(vinylidene chloride),poly(vinyl acetate), poly(ethylene glycol), polystyrene, polyisoprene,polyisobutylenes, poly(vinyl chloride), poly(propylene), poly(lacticacid), polyisocyanates, polycarbonates, alkyds, phenolics, epoxy resins,polysulfides, polyimides, liquid crystal polymers, heterocyclicpolymers, polypeptides, conducting polymers including polyacetylene,polyquinoline, polyaniline, polypyrrole, polythiophene, andpoly(p-phenylene), dendimers, fluoropolymers, derivatives thereof,combinations thereof.

In some embodiments, the particle includes a biodegradable polymer. Inother embodiments, the polymer is modified to be a biodegradable polymer(e.g., a poly(ethylene glycol) that is functionalized with a disulfidegroup). In some embodiments, the biodegradable polymer includes, withoutlimitation, one or more of a polyester, a polyanhydride, a polyamide, aphosphorous-based polymer, a poly(cyanoacrylate), a polyurethane, apolyorthoester, a polydihydropyran, a polyacetal, combinations thereof,or the like.

In some embodiments, the polyester includes, without limitation, one ormore of polylactic acid, polyglycolic acid, poly(hydroxybutyrate),poly(c-caprolactone), poly(β-malic acid), poly(dioxanones), combinationsthereof, or the like. In some embodiments, the polyanhydride includes,without limitation, one or more of poly(sebacic acid), poly(adipicacid), poly(terpthalic acid), combinations thereof, or the like. In yetother embodiments, the polyamide includes, without limitation, one ormore of poly(imino carbonates), polyaminoacids, combinations thereof, orthe like.

According to some embodiments, the phosphorous-based polymer includes,without limitation, one or more of a polyphosphate, a polyphosphonate, apolyphosphazene, combinations thereof, or the like. Further, in someembodiments, the biodegradable polymer further includes a polymer thatis responsive to a stimulus. In some embodiments, the stimulus includes,without limitation, one or more of pH, radiation, ionic strength,oxidation, reduction, temperature, an alternating magnetic field, analternating electric field, combinations thereof, or the like. In someembodiments, the stimulus includes an alternating magnetic field.

In still further embodiments, the material from which the particles areformed includes a non-wetting agent. According to another embodiment,the material is a liquid material in a single phase. In otherembodiments, the liquid material includes a plurality of phases. In someembodiments, the liquid material includes, without limitation, one ormore of multiple liquids, multiple immiscible liquids, surfactants,dispersions, emulsions, micro-emulsions, micelles, particulates,colloids, porogens, active ingredients, combinations thereof, or thelike.

According to other embodiments, the particle can be substantiallycoated. The coating, for example, can be a sugar based coating where thesugar is preferably a glucose, sucrose, maltose, derivatives thereof,combinations thereof, or the like.

Therapeutic Agent and/or Functionalization

In some embodiments, the particle includes a therapeutic or diagnosticagent coupled with the particle. The therapeutic or diagnostic agent canbe physically coupled or chemically coupled with the particle,encompassed within the particle, at least partially encompassed withinthe particle, coupled to the exterior of the particle, combinationsthereof, and the like. The therapeutic agent can be a drug, a biologic,a ligand, an oligopeptide, a cancer treating agent, a viral treatingagent, a bacterial treating agent, a fungal treating agent, combinationsthereof, or the like.

In some embodiments, a therapeutic agent for combination with theparticles of the presently disclosed subject matter is selected from oneof a drug and genetic material. In some embodiments, the geneticmaterial includes, without limitation, one or more of a non-viral genevector, DNA, RNA, RNAi, a viral particle, agents described elsewhereherein, combinations thereof, or the like.

In some embodiments, a pharmaceutical agent can be combined with theparticle material. The pharmaceutical agent can be, but is not limitedto, a drug, a peptide, RNAi, DNA, combinations thereof, or the like. Inother embodiments, the tag is selected from the group including afluorescence tag, a radiolabeled tag, a contrast agent, combinationsthereof, or the like. In some embodiments, the ligand includes a celltargeting peptide, or the like.

According to some embodiments, the particle is hydrophilic such that theparticle avoids clearance by biological organism, such as a human.

In yet other embodiments, the particle can include a functional locationsuch that the particle can be used as an analytical material. Accordingto such embodiments, a particle includes a functional molecular imprint.The functional molecular imprint can include functional monomersarranged as a negative image of a functional template. The functionaltemplate, for example, can be but is not limited to, chemicallyfunctional and size and shape equivalents of an enzyme, a protein, anantibiotic, an antigen, a nucleotide sequence, an amino acid, a drug, abiologic, nucleic acid, combinations thereof, or the like. In otherembodiments, the particle itself, for example, can be, but is notlimited to, an artificial functional molecule. In one embodiment, theartificial functional molecule is a functionalized particle that hasbeen molded from a molecular imprint. As such, a molecular imprint isgenerated in accordance with methods and materials of the presentlydisclosed subject matter and then a particle is formed from themolecular imprint, in accordance with further methods and materials ofthe presently disclosed subject matter. Such an artificial functionalmolecule includes substantially similar steric and chemical propertiesof a molecular imprint template. In one embodiment, the functionalmonomers of the functionalized particle are arranged substantially as anegative image of functional groups of the molecular imprint.

In some embodiments, additional components are included with thematerial of the particle to functionalize the particle. According tothese embodiments the additional components can be encased within theisolated structures, partially encased within the isolated structures,on the exterior surface of the isolated structures, combinationsthereof, or the like. Additional components can include, but are notlimited to, drugs, biologics, more than one drug, more than onebiologic, combinations thereof, and the like.

In some embodiments, the drug is a psychotherapeutic agent. In otherembodiments, the psychotherapeutic agent is used to treat depression andcan include, for example, sertraline, venlafaxine hydrochloride,paroxetine, bupropion, citalopram, fluoxetine, mirtazapine,escitalopram, and the like. In some embodiments, the psychotherapeuticagent is used to treat schizophrenia and can include, for example,olanazapine, risperidone, quetiapine, aripiprazole, ziprasidone, and thelike. According to other embodiments, the psychotherapeutic agent isused to treat attention deficit disorder (ADD) or attention deficithyperactivity disorder (ADHD), and can include, for example,methylphenidate, atomoxetine, amphetamine, dextroamphetamine, and thelike. In some other embodiments, the drug is a cholesterol drug and caninclude, for example, atorvastatin, simvastatin, pravastatin, ezetimibe,rosuvastatin, fenofibrate fluvastatin, and the like. In yet some otherembodiments, the drug is a cardiovascular drug and can include, forexample, amlodipine, valsartan, losartan, hydrochlorothiazide,metoprolol, candesartan, ramipril, irbesartan, amlodipine, benazepril,nifedipine, carvedilol, enalapril, telemisartan, quinapril, doxazosinmesylate, felodipine, lisinopril, and the like. In some embodiments, thedrug is a blood modifier and can include, for example, epoetin aifa,darbepoetin alfa, epoetin beta, clopidogrel, pegfilgrastim, filgrastim,enoxaparin, Factor VIIA, antihemophilic factor, immune globulin, and thelike. According to a further embodiment, the drug can include acombination of the above listed drugs.

In some embodiments, the material of the particles or the additionalcomponents included with the particles of the presently disclosedsubject matter can include, but are not limited, to anti-infectiveagents. In some embodiments, the anti-infective agent is used to treatbacterial infections and can include, for example, azithromycin,amoxicillin, clavulanic acid, levofloxacin, clarithromycin, ceftriaxone,ciprofloxacin, piperacillin, tazobactam sodium, imipenem, cilastatin,linezolid, meropenem, cefuroxime, moxitloxacin, and the like. In someembodiments the anti-infective agent is used to treat viral infectionsand can include, for example, lamivudine, zidovudine, valacyclovir,peginterferon, lopinavir, ritonavir, tenofovir, efavirenz, abacavir,lamivudine, zidovudine, atazanavir, and the like. In other embodiments,the anti-infective agent is used to treat fungal infections and caninclude, for example, terbinafine, fluconazole, itraconazole,caspofungin acetate, and the like. In some embodiments, the drug is agastrointestinal drug and can include, for example, esomeprazole,lansoprazole, omeprazole, pantoprazole, rabeprazole, ranitidine,ondansetron, and the like. According to yet other embodiments, the drugis a respiratory drug and can include, for example, fluticasone,salmeterol, montelukast, budesonide, formoterol, fexofenadine,cetirizine, desloratadine, mometasone furoate, tiotropium, albuterol,ipratropium, palivizumab, and the like. In yet other embodiments, thedrug is an antiarthritic drug and can include, for example, celecoxib,infliximab, etanercept, rofecoxib, valdecoxib, adalimumab, meloxicam,diclofenac, fentanyl, and the like. According to a further embodiment,the drug can include a combination of the above listed drugs.

According to alternative embodiments, the material of the particles orthe additional components included with the particles of the presentlydisclosed subject matter can include, but are not limited to ananticancer agent and can include, for example, nitrogen mustard,cisplatin, doxorubicin, docetaxel, anastrozole, trastuzumab,capecitabine, letrozole, leuprolide, bicalutamide, goserelin, rituximab,oxaliplatin, bevacizumab, irinotecan, paclitaxel, carboplatin, imatinib,gemcitabine, temozolomide, gefitinib, and the like. In some embodiments,the drug is a diabetes drug and can include, for example, rosiglitazone,pioglitazone, insulin, glimepiride, voglibose, and the like. In otherembodiments, the drug is an anticonvulsant and can include, for example,gabapentin, topiramate, oxcarbazepine, carbamazepine, lamotrigine,divalproex, levetiracetam, and the like. In some embodiments, the drugis a bone metabolism regulator and can include, for example,alendronate, raloxifene, risedronate, zoledronic, and the like. In someembodiments, the drug is a multiple sclerosis drug and can include, forexample, interferon, glatiramer, copolymer-1, and the like. In otherembodiments, the drug is a hormone and can include, for example,somatropin, norelgestromin, norethindrone, desogestrel, progestin,estrogen, octreotide, levothyroxine, and the like. In yet otherembodiments, the drug is a urinary tract agent, and can include, forexample, tamsulosin, finasteride, tolterodine, and the like. In someembodiments, the drug is an immunosuppressant and can include, forexample, mycophenolate mofetil, cyclosporine, tacrolimus, and the like.In some embodiments, the drug is an ophthalmic product and can include,for example, latanoprost, dorzolamide, botulinum, verteporfin, and thelike. In some embodiments, the drug is a vaccine and can include, forexample, pneumococcal, hepatitis, influenza, diphtheria, and the like.In other embodiments, the drug is a sedative and can include, forexample, zolpidem, zaleplon, eszopiclone, and the like. In someembodiments, the drug is an Alzheimer disease therapy and can include,for example, donepexil, rivastigmine, tacrine, and the like. In someembodiments, the drug is a sexual dysfunction therapy and can include,for example, sildenafil, tadalafil, alprostadil, levothyroxine, and thelike. In an alternative embodiment, the drug is an anesthetic and caninclude, for example, sevoflurane, propofol, mepivacaine, bupivacaine,ropivacaine, lidocaine, nesacaine, etidocaine, and the like. In someembodiments, the drug is a migraine drug and can include, for example,sumatriptan, almotriptan, rizatriptan, naratriptan, and the like. Insome embodiments, the drug is an infertility agent and can include, forexample, follitropin, choriogonadotropin, menotropin, folliclestimulating hormone (FSH), and the like. In some embodiments, the drugis a weight control product and can include, for example, orlistat,dexfenfluramine, sibutramine, and the like. According to a furtherembodiment, the drug can include a combination of the above listeddrugs. According to other embodiments, one or more other drugs can beincluded with the particles of the presently disclosed subject matterand can be found in Physician's Desk Reference, Thomson Healthcare, 59thBk&Cr edition (2004), which is incorporated herein by reference in itsentirety.

In some embodiments, one or more additional components are included withthe particles. The additional components can include: targeting ligandssuch as cell-targeting peptides, cell-penetrating peptides, integrinreceptor peptide (GRGDSP), melanocyte stimulating hormone, vasoactiveintestional peptide, anti-Her2 mouse antibodies and antibody fragments,and the like; vitamins; viruses; polysaccharides; cyclodextrins;liposomes; proteins; oligonucleotides; aptamers; optical nanoparticlessuch as CdSe for optical applications; borate nanoparticles to aid inboron neutron capture therapy (BNCT) targets; combinations thereof; andthe like.

In use, the particles of the presently disclosed subject matter can beused as treatment devices. In such uses, the particle is administered ina therapeutically effective amount to a patient.

Controlled or Timed Release

According to some embodiments, the particles can be controlled ortime-release drug delivery vehicles. A co-constituent of the particle,such as a polymer for example, can be cross-linked to varying degrees.Depending upon the amount of cross-linking of the polymer, anotherco-constituent of the particle, such as an active agent, can beconfigured to be released from the particle as desired. The active canbe released with no restraint, controlled release, or can be completelyrestrained within the particle. In some embodiments, the particle can befunctionalized, according to methods and materials disclosed herein, totarget a specific biological site, cell, tissue, agent, combinationsthereof, or the like. Upon interaction with the targeted biologicalstimulus, a co-constituent of the particle can be broken down to beginreleasing the active co-constituent of the particle. In one example, thepolymer can be poly(ethylene glycol) (PEG), which can be cross-linkedbetween about 5% and about 100%. The active co-constituent that can bedoxorubicin that is included in the cross-linked PEG particle. In oneembodiment, when the PEG co-constituent is cross-linked about 100%, nodoxorubicin leaches out of the particle.

In certain embodiments, the particle includes a composition of materialthat imparts controlled, delayed, immediate, or sustained release ofcargo of the particle or composition, such as for example, sustaineddrug release. According to some embodiments, materials and methods usedto form controlled, delayed, immediate, or sustained releasecharacteristics of the particles of the present invention include thematerials, methods, and formulations disclosed in U.S. PatentApplication nos. 2006/0099262; 2006/0104909; 200610110462; 2006/0127484;200410175428; 2004/0166157; and U.S. Pat. No. 6,964,780, each of whichare incorporated herein by reference in their entirety.

Particle Design

In some embodiments, the particle fabrication process provides controlof particle matrix composition, the ability for the particle to carry awide variety of cargos, the ability to functionalize the particle fortargeting and enhanced circulation, and/or the versatility to configurethe particle into different dosage forms, such as inhalation,dermatological, injectable, and oral, to name a few.

According to some embodiments, the matrix composition is tailored toprovide control over biocompatibility. In some embodiments, the matrixcomposition is tailored to provide control over cargo release. Thematrix composition, in some embodiments, contains biocompatiblematerials with solubility and/or philicity, controlled mesh density andcharge, stimulated degradation, and/or shape and size specificity whilemaintaining relative monodispersity.

According to further embodiments, the method for making particlescontaining cargo does not require the cargo to be chemically modified.In one embodiment, the method for producing particles is a gentleprocessing technique that allows for high cargo loading without the needfor covalent bonding. In one embodiment, cargo is physically entrappedwithin the particle due to interactions such as Van der Waals forces,electrostatic, hydrogen bonding, other other intra- and inter-molecularforces, combinations thereof, and the like.

In some embodiments, the particles are functionalized for targeting andenhanced circulation. In some embodiments, these features allow fortailored bioavailability. In one embodiment, the tailoredbioavailability increases delivery effectiveness. In one embodiment, thetailored bioavailability reduces side effects.

In some embodiments, a non-sperical particle has a surface area that isgreater than the surface area of spherical particle of the same volume.In some embodiments, the number of surface ligands on the particle isgreater than the number of surface ligands on a spherical particle ofthe same volume.

In some embodiments, one or more particles contain chemical moietyhandles for the attachment of protein. In some embodiments, the proteinis avidin. In some embodiments biotinylated reagents are subsequentlybound to the avidin. In some embodiments the protein is a cellpenetrating protein. In some embodiments, the protein is an antibodyfragment. In one embodiment, the particles are used for specifictargeting (e.g., breast tumors in female subjects). In some embodiments,the particles contain chemotherapeutics. In some embodiments, theparticles are composed of a cross link density or mesh density designedto allow slow release of the chemotherapeutic. The term crosslinkdensity means the mole fraction of prepolymer units that are crosslinkpoints. Prepolymer units include monomers, macromonomers and the like.

In some embodiments, the physical properties of the particle are variedto enhance cellular uptake. In some embodiments, the size (e.g., mass,volume, length or other geometric dimension) of the particle is variedto enhance cellular uptake. In some embodiments, the charge of theparticle is varied to enhance cellular uptake. In some embodiments, thecharge of the particle ligand is varied to enhance cellular uptake. Insome embodiments, the shape of the particle is varied to enhancecellular uptake.

In some embodiments, the physical properties of the particle are variedto enhance biodistribution. In some embodiments, the size (e.g., mass,volume, length or other geometric dimension) of the particle is variedto enhance biodistribution. In some embodiments, the charge of theparticle matrix is varied to enhance biodistribution. In someembodiments, the charge of the particle ligand is varied to enhancebiodistribution. In some embodiments, the shape of the particle isvaried to enhance biodistribution. In some embodiments, the aspect ratioof the particles is varied to enhance biodistribution.

In some embodiments, the physical properties of the particle are variedto enhance cellular adhesion. In some embodiments, the size (e.g., mass,volume, length or other geometric dimension) of the particle is variedto enhance cellular adhesion. In some embodiments, the charge of theparticle matrix is varied to enhance cellular adhesion. In someembodiments, the charge of the particle ligand is varied to enhancecellular adhesion. In some embodiments, the shape of the particle isvaried to enhance cellular adhesion.

In some embodiments, the particles are configured to degrade in thepresence of an intercellular stimulus. In some embodiments, theparticles are configured to degrade in a reducing environment. In someembodiments, the particles contain crosslinking agents that areconfigured to degrade in the presence of an external stimulus. In someembodiments, the crosslinking agents are configured to degrade in thepresence of a pH condition, a radiation condition, an ionic strengthcondition, an oxidation condition, a reduction condition, a temperaturecondition, an alternating magnetic field condition, an alternatingelectric field condition, combinations thereof, or the like. In someembodiments, the particles contain crosslinking agents that areconfigured to degrade in the presence of an external stimulus and/or atherapeutic agent.

In some embodiments, the particles contain crosslinking agents that areconfigured to degrade in the presence of an external stimulus, atargeting ligand, and a therapeutic agent. In some embodiments, thetherapeutic agent is a drug or a biologic. In some embodiments thetherapeutic agent is DNA, RNA, or siRNA.

In some embodiments, particles are configured to degrade in thecytoplasm of a cell. In some embodiments, particles are configured todegrade in the cytoplasm of a cell and release a therapeutic agent. Insome embodiments, the therapeutic agent is a drug or a biologic. In someembodiments the therapeutic agent is DNA, RNA, or siRNA. In someembodiments, the particles contain poly(ethylene glycol) andcrosslinking agents that degrade in the presence of an externalstimulus.

In some embodiments, the particles are used for ultrasound imaging. Insome embodiments, the particles used for ultrasound imaging are composedof bioabsorbable polymers. In some embodiments, particles used forultrasound imaging are porous. In some embodiments, particles used forultrasound imaging are composed of poly(lactic acid), poly(D,L-lacticacid-co-glycolic acid), and combinations thereof.

In some embodiments, the particles contain magnetite and are used ascontrast agents. In some embodiments, the particles contain magnetiteand are functionalized with linker groups and are used as contrastagents. In some embodiments, the particles, are functionalized with aprotein. In some embodiments, the particles are functionalized withN-hydroxysuccinimidyl ester groups. In some embodiments, avidin is boundto the particles. In some embodiments, particles containing magnetiteare covalently bound to avidin and exposed to a biotinylated reagent.

Particles to Mimic Natural Structures

In some embodiments, the particles are shaped to mimic naturalstructures. In some embodiments, the particles are shaped to mimicnatural structures and contain a therapeutic agent, a contrast agent, atargeting ligand, combination thereof, and the like.

Particles, materials of particles, and methods of making particles, makeit feasible to study physiological questions about the restriction ofparticle movement in the interstitial and perivascular space in brain.The size range and shape capabilities of the presently disclosedparticles increase the likelihood of gathering this information.

Mechano-chemico functionality plays a role in the bio-distribution andintracellular trafficking of carriers, such as the particles describedherein. Designing specific bio-distribution and intracellulartrafficking carries can make for a more efficacious diagnosis, and canlead to superior therapeutic and prevention strategies to fight disease.These specifically designed carries and methods can be extended to mimicbiological systems and, to manipulate biological systems.

Referring to FIG. 5, mammalian red blood cells are composed of a lipidmembrane coupled to a flexible cytoskeleton. The disc shape of red bloodcells provides a large surface-to-volume ratio which may aid inabsorption or exchange of oxygen and carbon dioxide. Red blood cells canpass through capillaries as small as 3 μm, having reversible elasticdeformation with strains less than 100%. Red blood cells severely deformthrough Intercellular gaps of sinusoids in the spleen, where stiffenedand aged red blood cells are removed after 120 days.

Enhanced rigidification of red blood cells is a key feature of thebiology and pathophysiology of malaria (Miller, et al. Nature (2002);Cooke, et al. Adv. Parasitology (2001); and Glenister, et. al Blood(2002); each of which is incorporated herein by reference). Sickle cellanemia is another RBC-based condition which is caused by elogated RBCs.PEGylation of RBCs has been used to camouflage the blood group antigensto make universal RBCs (Nacharaju et al. Transfusion (2005), which isincorporated herein by reference).

Referring to FIG. 6, in some embodiments, particles 8110 aresubstantially cell-shaped. In some embodiments, particles 8110 aresubstantially red blood cell-shaped. In some embodiments, particles 8110are substantially red blood cell-shaped and composed of a matrix with amodulus less than about 1 MPa. In some embodiments, the modulus can beselected from, for example, the modulus of elasticity, Young's modulus,or the like. In some embodiments, particles 8110 are substantially discshaped.

According to some embodiments, particles 8110 have dimensionssubstantially similar to red blood cells. In some embodiments, particle8110 has a largest linear dimension of about 5 μm to about 10 μm;preferably about 8 μm.

According to some embodiments, particle 8110 has a surface area of about130 μm². According to some embodiments, particle 8110 has a volume ofabout 98 μm³.

According to some embodiments, particles 8110 within a given pluralityof particles are monodisperse as described herein.

According to some embodiments, particle 8110 is configured to mimic theproperties of a red blood cell. According to some embodiments, particle8110 has a modulus of less than about 1 MPa. In some embodiments,particle 8110 has a modulus of less than about 1 MPa such that particle8110 can pass through a tube having an inner diameter of less than about3 μm. In some embodiments, the tube may be a blood vessel. The modulusof particle 8110 may be varied using porogens to create pores inparticle 8110.

According to some embodiments, particle 8110 includes polyethyleneglycol. In some embodiments, particle 8110 includesperfluororopolyether. In some embodiments, particle 8110 is hydrophilic.In some embodiments, the blood gas permeability of particle 8110 issubstantially similar to a red blood cell. In some embodiments, particle8110 comprises a material with high oxygen permeability.

Referring to FIG. 6, particle 8110 may have a surface functionalityand/or cargo. In some embodiments, the surface functionality and/orcargo is added to particle 8110 for a therapeutic or imaging purpose.Surface functionality and/or cargo may include petides, proteins,enzymes, imaging agents, linker groups, oliogos, siRNA, plasmid DNA,antigens or antibodies, viruses, killed bacteria, organelles, andtherapeutics. See also FIG. 7.

In some embodiments, particle 8110 includes surface functionality. Thesurface functionalization may include, for example, a natural mimic forextended delivery or imaging, PEGylation, or blood type antigens.

In some embodiments, particle 8110 includes cargo 8120. In someembodiments, cargo 8120 is capable of binding and/or releasing oxygen.In some embodiments, cargo 8120 is capable of binding and/or releasingcarbon dioxide. In some embodiments, cargo 8120 is capable of bindingand/or releasing oxygen and carbon dioxide. Cargo 8120 may include, butis not limited to, a therapeutic agent, hemoglobin, or an imaging agent.In some embodiments, cargo 8120 comprises a surface functionality. Inother embodiments, cargo 8120 is distributed throughout particle 8110.In some embodiments, cargo 8120 is on a surface of particle 8120.

In some embodiments, particle 8110 is loaded with mitochondria. Relevantproperties and uses of mitochondria are described in “Mitochondrial DNAIsolation Kit”, BioVision Research Products, available athttp://www.blovision.com/pdf/K280-50.pdf, which is incorporated byreference in its entirety. Mitochondria are known to aid in aerobicrespiration, the creation of the eukaryotic cell, and eventually complexmulticellular organisms. Recent reports have found that mitochondriaplay essential roles in aging and determining lifespan. A variety ofheritable and acquired diseases have been linked to mitochondrialdysfunction. In the article, “Mitochondrial Transfer Between Cells CanRescue Aerobic Respiration,” Tulane University Health Sciences Center,PNAS, vol. 102, no. 5, Jan. 31, 2006, available athttp://www.pnas.org/cgi/content/full/103/5/1283;http://www.futurepundit.com/mt/mt-altcomments.cgi?entry_id=2396, whichis incorporated by reference in its entirety, Jeffrey L. Spees et al.report that mitochondria are more dynamic than previously considered:mitochondria or mtDNA can move between cells. The active transfer fromadult stem cells and somatic cells can rescue aerobic respiration inmammalian cells with nonfunctional mitochondria.

Bid is a BH3 only member of the Bcl-2 family that regulates cell deathat the level of mitochondrial membranes. Bid appears to link themitochondrial pathway with the death receptor mediated pathway of celldeath. It is generally assumed that the f.l. (fulllength) proteinbecomes activated after proteolytic cleavage, especially by apicalcaspases like caspase 8. The cleaved protein then relocates tomitochondria and promotes membrane permeabilization, presumably byinteraction with mitochondrial lipids and other Bcl-2 proteins thatfacilitate the release of apoptogenic proteins like cytochrome c.Although the major action may reside in the C terminus part, tBid(cleaved Bid), un-cleaved Bid also has pro apoptotic potential whenectopically expressed in cells or in vitro. This pro apoptotic action offl. Bid has remained unexplained, especially at the biochemical level.In the article, “Pro-apoptotic Bid Induces Membrane Perturbation byInserting Selected Lysolipids into the Biolayer,” The University ofManchetser, Biochem. J., 387, pp. 109-118, 2005, available athttp://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1134938, whichis incorporated by reference in its entirety, Goonesinghe et al. explainthat f.l. (full-length) Bid can insert specific lysolipids into themembrane surface, thereby priming mitochondria for the release ofapoptogenic factors. This is reported to be most effective forlysophosphatidylcholine species to accumulate in mitochondria duringapoptosis induction. A Bid mutant that is not pro-apoptotic in vivo isdefective in lysophosphatidylcholine mediated membrane perturbation invitro. Results of this study provide a biochemical explanation for thepro-apoptotic action of f.l. Bid.

In some embodiments, particle 8110 is used to trigger cell death.Particle 8110 may be configured to trigger cell death by a variety ofmeans. According to some embodiments, cell death may be triggered by theincorporation of Cytochrome C into particle 8110 to trigger apoptosis bycombining with Apaf-1 (Tang, et. al Cell 2006), membrane attack complex(MAC) or tear/saliva enzymes to punch holes in cells, salts, andtherapeutics such as doxorubicin.

In some embodiments, the particles 8110 are configured to elicit animmune response. In some embodiments, particles 8110 are configured toelicit an immune response by at least one of the following methods:delivering antibodies for temporary immune protection inlong-circulating carriers; delivering monokines and lymphokines toregulate immune response; using antigens to trigger immunologicalresponse; and the delivery of viruses.

Referring to FIG. 8, particles 8110 may include epitope replicas:

T-independent response TIR: In some embodiments, particles 8110 evoke anantibody response. In some embodiments, particles 8110 stimulate B-cellsB directly.

T-dependent response TDR: In some embodiments, particles 8110 aretargeted to antigen-presenting cells APC (for example, dendritic cells)that are taken up via receptor mediated endocytosis. In someembodiments, bone marrow derived dendritic cells can be treated withparticles of varying cargos/ligands and measure their uptake and antigenpresenting competence.

In some embodiments, particles 8110 are configured to stimulate B-cellsB. In some embodiments, B-cells B are stimulated by targeting ligandscovalently bound to particles 8110. In some embodiments, B-cells B arestimulated by haptens bound to particles 8110. In some embodiments, theB-cells are stimulated by antigens bound to particles 8110.

Referring to FIG. 9, particles 8110 of FIG. 6 can also be used toreadily deliver organelles.

In some embodiments, particles 8110 are functionalized with targetingligands. In some embodiments, particles 8110 are functionalized totarget tumors. In some embodiments, particles 8110 are functionalized totarget breast tumors. In some embodiments, particles 8110 arefunctionalized to target the HER2 receptor. In some embodiments,particles 8110 are functionalized to target breast tumors and contain achemotherapeutic. In some embodiments, particles 8110 are functionalizedto target dendritic cells.

According to some embodiments, particles 8110 have a predeterminedzeta-potential.

IB. Introduction of Particle Precursor to Patterned Templates

According to some embodiments, the recesses of the patterned templatescan be configured to receive a substance to be molded. According to suchembodiments, variables such as, for example, the surface energy of thepatterned template, the volume of the recess, the permeability of thepatterned template, the viscosity of the substance to be molded as wellas other physical and chemical properties of the substance to be moldedinteract and affect the willingness of the recess to receive thesubstance to be molded.

II. Functionalization of Particles

In some embodiments, the presently disclosed subject matter provides amethod for functionalizing isolated micro- and/or nanoparticles.

In one embodiment, the functionalization includes introducing chemicalfunctional groups to a surface either physically or chemically. In someembodiments, the method of functionalization includes introducing atleast one chemical functional group to at least a portion ofmicroparticles and/or nanoparticles. In some embodiments, particles 3605are at least partially functionalized while particles 3605 are incontact with an article 3600. In one embodiment, the particles 3605 tobe functionalized are located within a mold or patterned template 108(FIGS. 10A-11D). In some embodiments, particles 3605 to befunctionalized are attached to a substrate (e.g., substrate 4010 ofFIGS. 12A-12D). In some embodiments, at least a portion of the exteriorof the particles 3605 can be chemically modified by performing the stepsillustrated in FIGS. 11A-11D. In one embodiment, the particles 3605 tobe functionalized are located within article 3600 as illustrated inFIGS. 11A and 12A. As illustrated in FIGS. 11A-11D and 12A-12D, someembodiments include contacting an article 3600 containing particles 3605with a solution 3602 containing a modifying agent 3604.

In one embodiment, illustrated in FIGS. 11C and 12C, modifying agent3604 attaches (e.g., chemically) to exposed particle surface 3606 bychemically reacting with or physically adsorbing to a linker group onparticle surface 3606. In one embodiment, the linker group on particle3606 is a chemical functional group that can attach to other species viachemical bond formation or physical affinity. In some embodiments,modifying agents 3611 are contained within or partially within particles3605. In some embodiments, the linker group includes a functional groupthat includes, without limitation, sulfides, amines, carboxylic acids,acid chlorides, alcohols, alkenes, alkyl halides, isocyanates, compoundsdisclosed elsewhere herein, combinations thereof, or the like.

In one embodiment, illustrated in FIGS. 11D and 12D, excess solution isremoved from article 3600 while particle 3605 remains in communicationwith article 3600. In some embodiments, excess solution is removed fromthe surface containing the particles. In some embodiments, excesssolution is removed by rinsing with or soaking in a liquid, by applyingan air stream, or by physically shaking or scraping the surface. In someembodiments, the modifying agent includes an agent selected from thegroup including dyes, fluorescent tags, radiolabeled tags, contrastagents, ligands, peptides, pharmaceutical agents, proteins, DNA, RNA,siRNA, compounds and materials disclosed elsewhere herein, combinationsthereof, and the like.

In one embodiment, functionalized particles 3608, 4008 are harvestedfrom article 3600 using, for example, methods described herein. In someembodiments, functionalizing and subsequently harvesting particles thatreside on an article (e.g., a substrate, a mold or patterned template)have advantages over other methods (e.g., methods in which the particlesmust be functionalized while in solution). In one embodiment of thepresently disclosed subject matter, fewer particles are lost in theprocess, giving a high product yield. In one embodiment of the presentlydisclosed subject matter, a more concentrated solution of the modifyingagent can be applied in lower volumes. In one embodiment of thepresently disclosed subject matter, where particles are functionalizedwhile they remain associated with article 3600, functionalization doesnot need to occur in a dilute solution. In one embodiment, the use ofmore concentrated solution facilitates, for example, the use of lowervolumes of modifying agent and/or lower times to functionalize.According to another embodiment, the functionalized particles areuniformly functionalized and each has substantially an identicalphysical load. In some embodiments, particles in a tight, 2-dimensionalarray, but not touching, are susceptible to application of thin,concentrated solutions for faster functionalization. In someembodiments, lower volume/higher concentration modifying agent solutionsare useful, for example, in connection with modifying agents that aredifficult and expensive to make and handle (e.g., biological agents suchas peptides, DNA, or RNA). In some embodiments, functionalizingparticles that remain connected to article 3600 eliminates difficultand/or time-consuming steps to remove excess unreacted material (e.g.,dialysis, extraction, filtration and column separation). In oneembodiment of the presently disclosed subject matter, highly purefunctionalized product can be produced at a reduced effort and cost.Because the particles are molded in a substantially inert polymer mold,the contents of the particle can be controlled, thereby yielding ahighly pure (e.g., greater than 95%) functionalized product.

In some embodiments, the liquid material from which the particles willbe formed, or particle precursor, is selected from the group including apolymer, a solution, a monomer, a plurality of monomers, apolymerization initiator, a polymerization catalyst, an inorganicprecursor, an organic material, a natural product, a metal precursor, apharmaceutical agent, a tag, a magnetic material, a paramagneticmaterial, a superparamagnetic material, a ligand, a cell penetratingpeptide, a porogen, a surfactant, a plurality of immiscible liquids, asolvent, a pharmaceutical agent with a binder, a charged species,combinations thereof, and the like. In some embodiments, thepharmaceutical agent Is selected from the group including a drug, apeptide, RNAi, DNA, combinations thereof, and the like. In someembodiments, the tag is selected from the group including a fluorescencetag, a radiolabeled tag, a contrast agent, combinations thereof, and thelike. In some embodiments, the ligand includes a cell targeting peptide.

III. Removing/Harvesting the Patterned Structures from the PatternedTemplate and/or Substrate

In some embodiments, the patterned structure (e.g., a patterned micro-or nanostructure) is removed from at least one of the patterned templateand/or the substrate. This can be accomplished by a number ofapproaches, including but not limited to applying the surface elementcontaining the patterned structure to a surface that has an affinity forthe patterned structure; applying the surface element containing thepatterned structure to a material that when hardened has a chemicaland/or physical interaction with the patterned structure; deforming thesurface element containing the patterned structure such that thepatterned structure is released from the surface element; swelling thesurface element containing the patterned structure with a first solventto extrude the patterned structure; and washing the surface elementcontaining the patterned structure with a second solvent that has anaffinity for the patterned structure.

In some embodiments, a surface has an affinity for the particles. Theaffinity of the surface can be a result of, in some embodiments, anadhesive or sticky surface, such as for example but not limitation,carbohydrates, epoxies, waxes, polyvinyl alcohol, polyvinyl pyrrolidone,polybutyl acrylate, polycyano acrylates, polyhydroxyethyl methacrylate,polymethyl methacrylate, combinations thereof, and the like. In someembodiments, the liquid is water that is cooled to form ice. In someembodiments, the water is cooled to a temperature below the Tm of waterbut above the Tg of the particle. In some embodiments the water iscooled to a temperature below the Tg of the particles but above the Tgof the mold or substrate. In some embodiments, the water is cooled to atemperature below the Tg of the mold or substrate.

In some embodiments, the first solvent includes supercritical fluidcarbon dioxide. In some embodiments, the first solvent includes water.In some embodiments, the first solvent includes an aqueous solutionincluding water and a detergent. In embodiments, the deforming thesurface element is performed by applying a mechanical force to thesurface element. In some embodiments, the method of removing thepatterned structure further includes a sonication method.

IV. Method of Patterning Natural and Synthetic Structures

In some embodiments, the presently disclosed subject matter describesmethods and processes, and products by processes, for generatingsurfaces and molds from natural structures, single molecules, orself-assembled structures. Accordingly, in some embodiments, thepresently disclosed subject matter describes a method of patterning anatural structure, single molecule, and/or a self-assembled structure.In some embodiments, the method further includes replicating the naturalstructure, single molecule, and/or a self-assembled structure. In someembodiments, the method further includes replicating the functionalityof the natural structure, single molecule, and/or a self-assembledstructure.

More particularly, in some embodiments, the method further includestaking the impression or mold of a natural structure, single molecule,and/or a self-assembled structure. In some embodiments, the impressionor mold is taken with a low surface energy polymeric precursor. In someembodiments, the low surface energy polymeric precursor includes aperfluoropolyether (PFPE) functionally terminated diacrylate. In someembodiments, the natural structure, single molecule, and/orself-assembled structure includes, without limitation, one or more ofenzymes, viruses, antibodies, micelles, tissue surfaces, combinationsthereof, or the like.

In some embodiments, the impression or mold is used to replicate thefeatures of the natural structure, single molecule, and/or aself-assembled structure into an isolated object or a surface. In someembodiments, a non-wetting imprint lithography method is used to impartthe features into a molded part or surface. In some embodiments, themolded part or surface produced by this process can be used in manyapplications, including, but not limited to, drug delivery, medicaldevices, coatings, catalysts, or mimics of the natural structures fromwhich they are derived. In some embodiments, the natural structureincludes biological tissue. In some embodiments, the biological tissueincludes tissue from a bodily organ, such as a heart. In someembodiments, the biological tissue includes vessels and bone. In someembodiments, the biological tissue includes tendon or cartilage. Forexample, in some embodiments, the presently disclosed subject matter canbe used to pattern surfaces for tendon and cartilage repair. Such repairtypically requires the use of collagen tissue, which comes from cadaversand must be machined for use as replacements. Most of these replacementsfail because one cannot lay down the primary pattern that is requiredfor replacement. The soft lithographic methods described hereinalleviate this problem.

In some embodiments, the presently disclosed subject matter can beapplied to tissue regeneration using stem cells. Almost all stem cellapproaches known in the art require molecular patterns for the cells toseed and then grow, thereby taking the shape of an organ, such as aliver, a kidney, or the like. In some embodiments, the molecularscaffold is cast and used as crystals to seed an organ in a form oftransplant therapy. In some embodiments, the stem cell andnano-substrate is seeded into a dying tissue, e.g., liver tissue, topromote growth and tissue regeneration. In some embodiments, thematerial to be replicated in the mold includes a material that issimilar to or the same as the material that was originally molded. Insome embodiments, the material to be replicated in the mold includes amaterial that is different from and/or has different properties than thematerial that was originally molded. This approach could play animportant role in addressing the organ transplant shortage.

In some embodiments, the presently disclosed subject matter is used totake the impression of one of an enzyme, a bacterium, and a virus. Insome embodiments, the enzyme, bacterium, or virus is then replicatedinto a discrete object or onto a surface that has the shape reminiscentof that particular enzyme, bacterium, or virus replicated into it. Insome embodiments, the mold itself is replicated on a surface, whereinthe surface-attached replicated mold acts as a receptor site for anenzyme, bacterium, or virus particle. In some embodiments, thereplicated mold is useful as a catalyst, a diagnostic sensor, atherapeutic agent, a vaccine, combinations thereof, and the like. Insome embodiments, the surface-attached replicated mold is used tofacilitate the discovery of new therapeutic agents.

In some embodiments, the macromolecular, e.g., enzyme, bacterial, orviral, molded “mimics” serve as non-self-replicating entities that havethe same surface topography as the original macromolecule, bacterium, orvirus. In some embodiments, the molded mimics are used to createbiological responses, e.g., an allergic response, to their presence,thereby creating antibodies or activating receptors. In someembodiments, the molded mimics function as a vaccine. In someembodiments, the efficacy of the biologically-active shape of the moldedmimics is enhanced by a surface modification technique.

According to embodiments of the present invention, a substance disclosedherein, for example, a drug, DNA, RNA, a biological molecule, a superabsorptive material, combinations thereof, and the like can be asubstance that is deposited into recesses and molded into particle 8110.According to still further embodiments, a substance to be molded is, butis not limited to, a polymer, a solution, a monomer, a plurality ofmonomers, a polymerization initiator, a polymerization catalyst, aninorganic precursor, a metal precursor, a pharmaceutical agent, a tag, amagnetic material, a paramagnetic material, a ligand, a cell penetratingpeptide, a porogen, a surfactant, a plurality of immiscible liquids, asolvent, a charged species, combinations thereof, and the like. In stillfurther embodiments, particle 8110 is, but is not limited to, organicpolymers, charged particles, polymer electrets (poly(vinylidenefluoride), Teflon-fluorinated ethylene propylene,polytetrafluoroethylene), therapeutic agents, drugs, non-viral genevectors, RNAi, viral particles, polymorphs, combinations thereof, andthe like.

EXAMPLES

The following Examples have been included to provide guidance to one ofordinary skill in the art for practicing representative embodiments ofthe presently disclosed subject matter. In light of the presentdisclosure and the general level of skill in the art, those of skill canappreciate that the following Examples are intended to be exemplary onlyand that numerous changes, modifications, and alterations can beemployed without departing from the scope of the presently disclosedsubject matter.

Example 1 Representative Procedure for Synthesis and Curing PhotocurablePerfluoropolyethers

In some embodiments, the synthesis and curing of PFPE materials of thepresently disclosed subject matter is performed by using the methoddescribed by Rolland, J. P., et al., J. Am. Chem. Soc., 2004, 126,2322-2323. Briefly, this method involves themethacrylate-functionalization of a commercially available PFPE diol(M_(n)=3800 g/mol) with isocyanatoethyl methacrylate. Subsequentphotocuring of the material is accomplished through blending with 1 wt %of 2,2-dimethoxy-2-phenylacetophenone and exposure to UV radiation(λ=365 nm).

More particularly, in a typical preparation of perfluoropolyetherdimethacrylate (PFPE DMA), poly(tetrafluoroethyleneoxide-co-difluoromethylene oxide)α,ω diol (ZDOL, average M_(n) ca. 3,800g/mol, 95%, Aldrich Chemical Company, Milwaukee, Wis., United States ofAmerica) (5.7227 g, 1.5 mmol) was added to a dry 50 mL round bottomflask and purged with argon for 15 minutes. 2-isocyanatoethylmethacrylate (EIM, 99%, Aldrich) (0.43 mL, 3.0 mmol) was then added viasyringe along with 1,1,2-trichlorotrifluoroethane (Freon 113 99%,Aldrich) (2 mL), and dibutyltin diacetate (DBTDA, 99%, Aldrich) (50 μL).The solution was immersed in an oil bath and allowed to stir at 50° C.for 24 h. The solution was then passed through a chromatographic column(alumina, Freon 113, 2×5 cm). Evaporation of the solvent yielded aclear, colorless, viscous oil, which was further purified by passagethrough a 0.22-μm polyethersulfone filter.

In a representative curing procedure for PFPE DMA, 1 wt % of2,2-dimethoxy-2-phenyl acetophenone (DMPA, 99% Aldrich), (0.05 g, 2.0mmol) was added to PFPE DMA (5 g, 1.2 mmol) along with 2 mL Freon 113until a clear solution was formed. After removal of the solvent, thecloudy viscous oil was passed through a 0.22-μm polyethersulfone filterto remove any DMPA that did not disperse into the PFPE DMA. The filteredPFPE DMA was then irradiated with a UV source (Electro-Lite Corporation,Danbury, Conn., United States of America, UV curing chamber model no.81432-ELC-500, λ=365 nm) while under a nitrogen purge for 10 min. Thisresulted in a clear, slightly yellow, rubbery material.

Example 2 Representative Fabrication of a PFPE DMA Device

In some embodiments, a PFPE DMA device, such as a stamp, was fabricatedaccording to the method described by Rolland, J. P. et al., J. Am. Chem.Soc., 2004, 126, 2322-2323. Briefly, the PFPE DMA containing aphotoinitiator, such as DMPA, was spin coated (800 rpm) to a thicknessof 20 μm onto a Si wafer containing the desired photoresist pattern.This coated wafer was then placed into the UV curing chamber andirradiated for 6 seconds. Separately, a thick layer (about 5 mm) of thematerial was produced by pouring the PFPE DMA containing photoinitiatorinto a mold surrounding the Si wafer containing the desired photoresistpattern. This wafer was irradiated with UV light for one minute.Following this, the thick layer was removed. The thick layer was thenplaced on top of the thin layer such that the patterns in the two layerswere precisely aligned, and then the entire device was irradiated for 10minutes. Once complete, the entire device was peeled from the Si waferwith both layers adhered together.

Example 3 Fabrication of Isolated Particles Using Non-Wetting ImprintLithography

3.1 Fabrication of 200-nm Trapezoidal PEG Particles

A patterned perfluoropolyether (PFPE) mold is generated by pouring aPFPE-dimethacrylate (PFPE-DMA) containing 1-hydroxycyclohexyl phenylketone over a silicon substrate patterned with 200-nm trapezoidal shapes(See FIG. 13). A poly(dimethylsiloxane) mold is used to confine theliquid PFPE-DMA to the desired area. The apparatus was then subjected toUV light (λ=365 nm) for 10 minutes while under a nitrogen purge. Thefully cured PFPE-DMA mold was then released from the silicon master.Separately, a poly(ethylene glycol) (PEG) diacrylate (n=9) is blendedwith 1 wt % of a photoinitiator, 1-hydroxycyclohexyl phenyl ketone.Flat, uniform, non-wetting surfaces are generated by treating a siliconwafer cleaned with “piranha” solution (1:1 concentrated sulfuric acid:30% hydrogen peroxide (aq) solution) with trichloro(1H, 1H, 2H,2H-perfluorooctyl) silane via vapor deposition in a desiccator for 20minutes. Following this, 50 μL of PEG diacrylate is then placed on thetreated silicon wafer and the patterned PFPE mold placed on top of it.The substrate is then placed in a molding apparatus and a small pressureis applied to push out excess PEG-diacrylate. The pressure used was atleast about 100 N/cm^(z). The entire apparatus was then subjected to UVlight (λ=365 nm) for ten minutes while under a nitrogen purge. Particlesare observed after separation of the PFPE mold and the treated siliconwafer using scanning electron microscopy (SEM) (see FIG. 14).

3.2 Fabrication of 500-nm Conical PEG Particles

A patterned perfluoropolyether (PFPE) mold is generated by pouring aPFPE-dimethacrylate (PFPE-DMA) containing 1-hydroxycyclohexyl phenylketone over a silicon substrate patterned with 500-nm conical shapes(see FIG. 15). A poly(dimethylsiloxane) mold is used to confine theliquid PFPE-DMA to the desired area. The apparatus is then subjected toUV light (λ=365 nm) for 10 minutes while under a nitrogen purge. Thefully cured PFPE-DMA mold is then released from the silicon master.Separately, a poly(ethylene glycol) (PEG) diacrylate (n=9) is blendedwith 1 wt % of a photoinitiator, 1-hydroxycyclohexyl phenyl ketone.Flat, uniform, non-wetting surfaces are generated by treating a siliconwafer cleaned with “piranha” solution (1:1 concentrated sulfuric acid:30% hydrogen peroxide (aq) solution) with trichloro(1H, 1H, 2H,2H-perfluorooctyl) silane via vapor deposition in a desiccator for 20minutes. Following this, 50 μL of PEG diacrylate is then placed on thetreated silicon wafer and the patterned PFPE mold placed on top of it.The substrate is then placed in a molding apparatus and a small pressureis applied to push out excess PEG-diacrylate. The entire apparatus isthen subjected to UV light (λ=365 nm) for ten minutes while under anitrogen purge. Particles are observed after separation of the PFPE moldand the treated silicon wafer using scanning electron microscopy (SEM)(see FIG. 16).

3.3 Fabrication of 3-μm Arrow-Shaped PEG Particles

A patterned perfluoropolyether (PFPE) mold is generated by pouring aPFPE-dimethacrylate (PFPE-DMA) containing 1-hydroxycyclohexyl phenylketone over a silicon substrate patterned with 3-μm arrow shapes (seeFIG. 17). A poly(dimethylsiloxane) mold is used to confine the liquidPFPE-DMA to the desired area. The apparatus is then subjected to UVlight (λ=365 nm) for 10 minutes while under a nitrogen purge. The fullycured PFPE-DMA mold is then released from the silicon master.Separately, a poly(ethylene glycol) (PEG) diacrylate (n=9) is blendedwith 1 wt % of a photoinitiator, 1-hydroxycyclohexyl phenyl ketone.Flat, uniform, non-wetting surfaces are generated by treating a siliconwafer cleaned with “piranha” solution (1:1 concentrated sulfuric acid:30% hydrogen peroxide (aq) solution) with trichloro(1H, 1H, 2H,2H-perfluorooctyl) silane via vapor deposition in a desiccator for 20minutes. Following this, 50 μL of PEG diacrylate is then placed on thetreated silicon wafer and the patterned PFPE mold placed on top of it.The substrate is then placed in a molding apparatus and a small pressureis applied, to push out excess PEG-diacrylate. The entire apparatus isthen subjected to UV light (λ=365 nm) for ten minutes while under anitrogen purge. Particles are observed after separation of the PFPE moldand the treated silicon wafer using scanning electron microscopy (SEM)(see FIG. 18).

3.4 Fabrication of 200-nm×750-nm×250-nm Rectangular PEG Particles

A patterned perfluoropolyether (PFPE) mold is generated by pouring aPFPE-dimethacrylate (PFPE-DMA) containing 1-hydroxycyclohexyl phenylketone over a silicon substrate patterned with 200-nm×750-nm×250-nmrectangular shapes. A poly(dimethylsiloxane) mold is used to confine theliquid PFPE-DMA to the desired area. The apparatus is then subjected toUV light (λ=365 nm) for 10 minutes while under a nitrogen purge. Thefully cured PFPE-DMA mold is then released from the silicon master.Separately, a poly(ethylene glycol) (PEG) diacrylate (n=9) is blendedwith 1 wt % of a photoinitiator, 1-hydroxycyclohexyl phenyl ketone.Flat, uniform, non-wetting surfaces are generated by treating a siliconwafer cleaned with “piranha” solution (1:1 concentrated sulfuric acid:30% hydrogen peroxide (aq) solution) with trichloro(1H, 1H, 2H,2H-perfluorooctyl) silane via vapor deposition in a desiccator for 20minutes. Following this, 50 μL of PEG diacrylate is then placed on thetreated silicon wafer and the patterned PFPE mold placed on top of it.The substrate is then placed in a molding apparatus and a small pressureis applied to push out excess PEG-diacrylate. The entire apparatus isthen subjected to UV light (λ=365 nm) for ten minutes while under anitrogen purge. Particles are observed after separation of the PFPE moldand the treated silicon wafer using scanning electron microscopy (SEM)(see FIG. 19).

3.5 Fabrication of 200-nm Trapezoidal Trimethylopropane Triacrylate(TMPTA) Particles

A patterned perfluoropolyether (PFPE) mold is generated by pouring aPFPE-dimethacrylate (PFPE-DMA) containing 1-hydroxycyclohexyl phenylketone over a silicon substrate patterned with 200-nm trapezoidal shapes(see FIG. 13). A poly(dimethylsiloxane) mold is used to confine theliquid PFPE-DMA to the desired area. The apparatus is then subjected toUV light (λ=365 nm) for 10 minutes while under a nitrogen purge. Thefully cured PFPE-DMA mold is then released from the silicon master.Separately, TMPTA is blended with 1 wt % of a photoinitiator,1-hydroxycyclohexyl phenyl ketone. Flat, uniform, non-wetting surfacesare generated by treating a silicon wafer cleaned with “piranha”solution (1:1 concentrated sulfuric acid: 30% hydrogen peroxide (aq)solution) with trichloro(1H, 1H, 2H, 2H-perfluorooctyl) silane via vapordeposition in a desiccator for 20 minutes. Following this, 50 μL ofTMPTA is then placed on the treated silicon wafer and the patterned PFPEmold placed on top of it. The substrate is then placed in a moldingapparatus and a small pressure is applied to push out excess TMPTA. Theentire apparatus is then subjected to UV light (λ=365 nm) for tenminutes while under a nitrogen purge. Particles are observed afterseparation of the PFPE mold and the treated silicon wafer using scanningelectron microscopy (SEM) (see FIG. 20).

3.6 Fabrication of 500-nm Conical Trimethyloporopane Triacrylate (TMPTA)Particles

A patterned perfluoropolyether (PFPE) mold is generated by pouring aPFPE-dimethacrylate (PFPE-DMA) containing 1-hydroxycyclohexyl phenylketone over a silicon substrate patterned with 500-nm conical shapes(see FIG. 15). A poly(dimethylsiloxane) mold is used to confine theliquid PFPE-DMA to the desired area. The apparatus is then subjected toUV light (λ=365 nm) for 10 minutes while under a nitrogen purge. Thefully cured PFPE-DMA mold is then released from the silicon master.Separately, TMPTA is blended with 1 wt % of a photoinitiator,1-hydroxycyclohexyl phenyl ketone. Flat, uniform, non-wetting surfacesare generated by treating a silicon wafer cleaned with “piranha”solution (1:1 concentrated sulfuric acid: 30% hydrogen peroxide (aq)solution) with trichloro(1H, 1H, 2H, 2H-perfluorooctyl) silane via vapordeposition in a desiccator for 20 minutes. Following this, 50 μL ofTMPTA is then placed on the treated silicon wafer and the patterned PFPEmold placed on top of it. The substrate is then placed in a moldingapparatus and a small pressure is applied to push out excess TMPTA. Theentire apparatus is then subjected to UV light (λ=365 nm) for tenminutes while under a nitrogen purge. Particles are observed afterseparation of the PFPE mold and the treated silicon wafer using scanningelectron microscopy (SEM) (see FIG. 21). Further, FIG. 22 shows ascanning electron micrograph of 500-nm isolated conical particles ofTMPTA, which have been printed using an embodiment of the presentlydescribed non-wetting imprint lithography method and harvestedmechanically using a doctor blade. The ability to harvest particles insuch a way offers conclusive evidence for the absence of a “scum layer.”

3.7 Fabrication of 3-μm Arrow-Shaped TMPTA Particles

A patterned perfluoropolyether (PFPE) mold is generated by pouring aPFPE-dimethacrylate (PFPE-DMA) containing 1-hydroxycyclohexyl phenylketone over a silicon substrate patterned with 3-μm arrow shapes (seeFIG. 17). A poly(dimethylsiloxane) mold is used to confine the liquidPFPE-DMA to the desired area. The apparatus is then subjected to UVlight (λ=365 nm) for 10 minutes while under a nitrogen purge. The fullycured PFPE-DMA mold is then released from the silicon master.Separately, TMPTA is blended with 1 wt % of a photoinitiator,1-hydroxycyclohexyl phenyl ketone. Flat, uniform, non-wetting surfacesare generated by treating a silicon wafer cleaned with “piranha”solution (1:1 concentrated sulfuric acid: 30% hydrogen peroxide (aq)solution) with trichloro(1H, 1H, 2H, 2H-perfluorooctyl) silane via vapordeposition in a desiccator for 20 minutes. Following this, 50 μL ofTMPTA is then placed on the treated silicon wafer and the patterned PFPEmold placed on top of it. The substrate is then placed in a moldingapparatus and a small pressure is applied to push out excess TMPTA. Theentire apparatus is then subjected to UV light (λ=365 nm) for tenminutes while under a nitrogen purge. Particles are observed afterseparation of the PFPE mold and the treated silicon wafer using scanningelectron microscopy (SEM).

3.8 Fabrication of 200-nm Trapezoidal Poly(Lactic Acid) (PLA) Particles

A patterned perfluoropolyether (PFPE) mold is generated by pouring aPFPE-dimethacrylate (PFPE-DMA) containing 1-hydroxycyclohexyl phenylketone over a silicon substrate patterned with 200-nm trapezoidal shapes(see FIG. 13). A poly(dimethylsiloxane) mold is used to confine theliquid PFPE-DMA to the desired area. The apparatus is then subjected toUV light (λ=365 nm) for 10 minutes while under a nitrogen purge. Thefully cured PFPE-DMA mold is then released from the silicon master.Separately, one gram of (3S)-cis-3,6-dimethyl-1,4-dioxane-2,5-dione (LA)is heated above its melting temperature (92° C.) to 110° C. andapproximately 20 μL of stannous octoate catalystlinitiator is added tothe liquid monomer. Flat, uniform, non-wetting surfaces are generated bytreating a silicon wafer cleaned with “piranha” solution (1:1concentrated sulfuric acid: 30% hydrogen peroxide (aq) solution) withtrichloro(1H, 1H, 2H, 2H-perfluorooctyl) silane via vapor deposition ina desiccator for 20 minutes. Following this, 50 μL of molten LAcontaining catalyst is then placed on the treated silicon waferpreheated to 110° C. and the patterned PFPE mold is placed on top of it.The substrate is then placed in a molding apparatus and a small pressureis applied to push out excess monomer. The entire apparatus is thenplaced in an oven at 110° C. for 15 hours. Particles are observed aftercooling to room temperature and separation of the PFPE mold and thetreated silicon wafer using scanning electron microscopy (SEM) (see FIG.23). Further, FIG. 24 is a scanning electron micrograph of 200-nmisolated trapezoidal particles of poly(lactic acid) (PLA), which havebeen printed using an embodiment of the presently described non-wettingimprint lithography method and harvested mechanically using a doctorblade. The ability to harvest particles in such a way offers conclusiveevidence for the absence of a “scum layer.”

3.9 Fabrication of 3-μm Arrow-Shaped (PLA) Particles

A patterned perfluoropolyether (PFPE) mold is generated by pouring aPFPE-dimethacrylate (PFPE-DMA) containing 1-hydroxycyclohexyl phenylketone over a silicon substrate patterned with 3-μm arrow shapes (seeFIG. 17). A poly(dimethylsiloxane) mold is used to confine the liquidPFPE-DMA to the desired area. The apparatus is then subjected to UVlight (λ=365 nm) for 10 minutes while under a nitrogen purge. The fullycured PFPE-DMA mold is then released from the silicon master.Separately, one gram of (3S)-cis-3,6-dimethyl-1,4-dioxane-2,5-dione (LA)is heated above its melting temperature (92° C.) to 110° C. andapproximately 20 μL of stannous octoate catalyst/initiator is added tothe liquid monomer. Flat, uniform, non-wetting surfaces are generated bytreating a silicon wafer cleaned with “piranha” solution (1:1concentrated sulfuric acid: 30% hydrogen peroxide (aq) solution) withtrichloro(1H, 1H, 2H, 2H-perfluorooctyl) silane via vapor deposition ina desiccator for 20 minutes. Following this, 50 μL of molten LAcontaining catalyst is then placed on the treated silicon waferpreheated to 110° C. and the patterned PFPE mold is placed on top of it.The substrate is then placed in a molding apparatus and a small pressureis applied to push out excess monomer. The entire apparatus is thenplaced in an oven at 110° C. for 15 hours. Particles are observed aftercooling to room temperature and separation of the PFPE mold and thetreated silicon wafer using scanning electron microscopy (SEM) (see FIG.25).

3.10 Fabrication of 500-nm Conical Shaped (PLA Particles

A patterned perfluoropolyether (PFPE) mold is generated by pouring aPFPE-dimethacrylate (PFPE-DMA) containing 1-hydroxycyclohexyl phenylketone over a silicon substrate patterned with 500-nm conical shapes(see FIG. 15). A poly(dimethylsiloxane) mold is used to confine theliquid PFPE-DMA to the desired area. The apparatus is then subjected toUV light (λ=365 nm) for 10 minutes while under a nitrogen purge. Thefully cured PFPE-DMA mold is then released from the silicon master.Separately, one gram of (3S)-cis-3,6-dimethyl-1,4-dioxane-2,5-dione (LA)is heated above its melting temperature (92° C.) to 110° C. andapproximately 20 μL of stannous octoate catalyst/initiator is added tothe liquid monomer. Flat, uniform, non-wetting surfaces are generated bytreating a silicon wafer cleaned with “piranha” solution (1:1concentrated sulfuric acid: 30% hydrogen peroxide (aq) solution) withtrichloro(1H, 1H, 2H, 2H-perfluorooctyl) silane via vapor deposition ina desiccator for 20 minutes. Following this, 50 μL of molten LAcontaining catalyst is then placed on the treated silicon waferpreheated to 110° C. and the patterned PFPE mold is placed on top of it.The substrate is then placed in a molding apparatus and a small pressureis applied to push out excess monomer. The entire apparatus is thenplaced in an oven at 110° C. for 15 hours. Particles are observed aftercooling to room temperature and separation of the PFPE mold and thetreated silicon wafer using scanning electron microscopy (SEM) (see FIG.26).

3.11 Fabrication of 200-nm Trapezoidal Poly(Pyrrole) (Ppy) Particles

A patterned perfluoropolyether (PFPE) mold is generated by pouring aPFPE-dimethacrylate (PFPE-DMA) containing 1-hydroxycyclohexyl phenylketone over a silicon substrate patterned with 200-nm trapezoidal shapes(see FIG. 13). A poly(dimethylsiloxane) mold is used to confine theliquid PFPE-DMA to the desired area. The apparatus is then subjected toUV light (λ=365 nm) for 10 minutes while under a nitrogen purge. Thefully cured PFPE-DMA mold is then released from the silicon master.Flat, uniform, non-wetting surfaces are generated by treating a siliconwafer cleaned with “piranha” solution (1:1 concentrated sulfuric acid:30% hydrogen peroxide (aq) solution) with trichloro(1H, 1H, 2H,2H-perfluorooctyl) silane via vapor deposition in a desiccator for 20minutes. Separately, 50 μL of a 1:1 v:v solution oftetrahydrofuran:pyrrole is added to 50 μL of 70% perchloric acid (aq). Aclear, homogenous, brown solution quickly forms and develops into black,solid, polypyrrole in 15 minutes. A drop of this clear, brown solution(prior to complete polymerization) is placed onto a treated siliconwafer and into a stamping apparatus and a pressure is applied to removeexcess solution. The apparatus is then placed into a vacuum oven for 15h to remove the THF and water. Particles are observed using scanningelectron microscopy (SEM) (see FIG. 27) after release of the vacuum andseparation of the PFPE mold and the treated silicon wafer.

3.12 Fabrication of 3-μm Arrow-Shaped (Ppy) Particles

A patterned perfluoropolyether (PFPE) mold is generated by pouring aPFPE-dimethacrylate (PFPE-DMA) containing 1-hydroxycyclohexyl phenylketone over a silicon substrate patterned with 3-μm arrow shapes (seeFIG. 17). A poly(dimethylsiloxane) mold is used to confine the liquidPFPE-DMA to the desired area. The apparatus is then subjected to UVlight (λ=365 nm) for 10 minutes while under a nitrogen purge. The fullycured PFPE-DMA mold is then released from the silicon master. Flat,uniform, non-wetting surfaces are generated by treating a silicon wafercleaned with “piranha” solution (1:1 concentrated sulfuric acid: 30%hydrogen peroxide (aq) solution) with trichloro(1H, 1H, 2H,2H-perfluorooctyl) silane via vapor deposition in a desiccator for 20minutes. Separately, 50 μL of a 1:1 v:v solution oftetrahydrofuran:pyrrole is added to 50 μL of 70% perchloric acid (aq). Aclear, homogenous, brown solution quickly forms and develops into black,solid, polypyrrole in 15 minutes. A drop of this clear, brown solution(prior to complete polymerization) is placed onto a treated siliconwafer and into a stamping apparatus and a pressure is applied to removeexcess solution. The apparatus is then placed into a vacuum oven for 15h to remove the THF and water. Particles are observed using scanningelectron microscopy (SEM) (see FIG. 28) after release of the vacuum andseparation of the PFPE mold and the treated silicon wafer.

3.13 Fabrication of 500-nm Conical (Ppy) Particles

A patterned perfluoropolyether (PFPE) mold is generated by pouring aPFPE-dimethacrylate (PFPE-DMA) containing 1-hydroxycyclohexyl phenylketone over a silicon substrate patterned with 500-nm conical shapes(see FIG. 15). A poly(dimethylsiloxane) mold is used to confine theliquid PFPE-DMA to the desired area. The apparatus is then subjected toUV light (λ=365 nm) for 10 minutes while under a nitrogen purge. Thefully cured PFPE-DMA mold is then released from the silicon master.Flat, uniform, non-wetting surfaces are generated by treating a siliconwafer cleaned with “piranha” solution (1:1 concentrated sulfuric acid:30% hydrogen peroxide (aq) solution) with trichloro(1H, 1H, 2H,2H-perfluorooctyl) silane via vapor deposition in a desiccator for 20minutes. Separately, 50 μL of a 1:1 v:v solution oftetrahydrofuran:pyrrole is added to 50 μL of 70% perchloric acid (aq). Aclear, homogenous, brown solution quickly forms and develops into black,solid, polypyrrole in 15 minutes. A drop of this clear, brown solution(prior to complete polymerization) is placed onto a treated siliconwafer and into a stamping apparatus and a pressure is applied to removeexcess solution. The apparatus is then placed into a vacuum oven for 15h to remove the THF and water. Particles are observed using scanningelectron microscopy (SEM) (see FIG. 29) after release, of the vacuum andseparation of the PFPE mold and the treated silicon wafer.

3.14 Encapsulation of Fluorescently Tagged DNA Inside 200-nm TrapezoidalPEG Particles

A patterned perfluoropolyether (PFPE) mold is generated by pouring aPFPE-dimethacrylate (PFPE-DMA) containing 1-hydroxycyclohexyl phenylketone over a silicon substrate patterned with 200-nm trapezoidal shapes(see FIG. 13). A poly(dimethylsiloxane) mold is used to confine theliquid PFPE-DMA to the desired area. The apparatus is then subjected toUV light (λ=365 nm) for 10 minutes while under a nitrogen purge. Thefully cured PFPE-DMA mold is then released from the silicon master.Separately, a poly(ethylene glycol) (PEG) diacrylate (n=9) is blendedwith 1 wt % of a photoinitiator, 1-hydroxycyclohexyl phenyl ketone. 20μL of water and 20 μL of PEG diacrylate monomer are added to 8 nanomolesof 24 bp DNA oligonucleotide that has been tagged with a fluorescentdye, CY-3. Flat, uniform, non-wetting surfaces are generated by treatinga silicon wafer cleaned with “piranha” solution (1:1 concentratedsulfuric acid: 30% hydrogen peroxide (aq) solution) with trichloro(1H,1H, 2H, 2H-perfluorooctyl) silane via vapor deposition in a desiccatorfor 20 minutes. Following this, 50 μL of the PEG diacrylate solution isthen placed on the treated silicon wafer and the patterned PFPE moldplaced on top of it. The substrate is then placed in a molding apparatusand a small pressure is applied to push out excess PEG-diacrylatesolution. The entire apparatus is then subjected to UV light (λ=365 nm)for ten minutes while under a nitrogen purge. Particles are observedafter separation of the PFPE mold and the treated silicon wafer usingconfocal fluorescence microscopy (see FIG. 30). Further, FIG. 30A showsa fluorescent confocal micrograph of 200-nm trapezoidal PEGnanoparticles, which contain 24-mer DNA strands that are tagged withCY-3. FIG. 30B is optical micrograph of the 200-nm isolated trapezoidalparticles of PEG diacrylate that contain fluorescently tagged DNA. FIG.30C is the overlay of the images provided in FIGS. 30A and 30B, showingthat every particle contains DNA.

3.15 Encapsulation of Magnetite Nanoparticles Inside 500-nm Conical PEGParticles

A patterned perfluoropolyether (PFPE) mold is generated by pouring aPFPE-dimethacrylate (PFPE-DMA) containing 1-hydroxycyclohexyl phenylketone over a silicon substrate patterned with 500-nm conical shapes(see FIG. 15). A poly(dimethylsiloxane) mold is used to confine theliquid PFPE-DMA to the desired area. The apparatus is then subjected toUV light (λ=365 nm) for 10 minutes while under a nitrogen purge. Thefully cured PFPE-DMA mold is then released from the silicon master.Flat, uniform, non-wetting surfaces are generated by treating a siliconwafer cleaned with “piranha” solution (1:1 concentrated sulfuric acid:30% hydrogen peroxide (aq) solution) with trichloro(1H, 1H, 2H,2H-perfluorooctyl) silane via vapor deposition in a desiccator for 20minutes. Separately, citrate capped magnetite nanoparticles weresynthesized by reaction of ferric chloride (40 mL of a 1 M aqueoussolution) and ferrous chloride (10 mL of a 2 M aqueous hydrochloric acidsolution) which is added to ammonia (500 mL of a 0.7 M aqueoussolution). The resulting precipitate is collected by centrifugation andthen stirred in 2 M perchloric acid. The final solids are collected bycentrifugation. 0.290 g of these perchlorate-stabilized nanoparticlesare suspended in 50 mL of water and heated to 90° C. while stirring.Next, 0.106 g of sodium citrate is added. The solution is stirred at 90°C. for 30 min to yield an aqueous solution of citrate-stabilized ironoxide nanoparticles. 50 μL of this solution is added to 50 μL of a PEGdiacrylate solution in a microtube. This microtube is vortexed for tenseconds. Following this, 50 μL of this PEG diacrylate/particle solutionis then placed on the treated silicon wafer and the patterned PFPE moldplaced on top of it. The substrate is then placed in a molding apparatusand a small pressure is applied to push out excessPEG-diacrylate/particle solution. The entire apparatus is then subjectedto UV light (λ=365 nm) for ten minutes while under a nitrogen purge.Nanoparticle-containing PEG-diacrylate particles are observed afterseparation of the PFPE mold and the treated silicon wafer using opticalmicroscopy.

3.16 Fabrication of Isolated Particles on Glass Surfaces Using “Doublestamping”

A patterned perfluoropolyether (PFPE) mold is generated by pouring aPFPE-dimethacrylate (PFPE-DMA) containing 1-hydroxycyclohexyl phenylketone over a silicon substrate patterned with 200-nm trapezoidal shapes(see FIG. 13). A poly(dimethylsiloxane) mold is used to confine theliquid PFPE-DMA to the desired area. The apparatus is then subjected toUV light (λ=365 nm) for 10 minutes while under a nitrogen purge. Thefully cured PFPE-DMA mold is then released from the silicon master.Separately, a poly(ethylene glycol) (PEG) diacrylate (n=9) is blendedwith 1 wt % of a photoinitiator, 1-hydroxycyclohexyl phenyl ketone. Aflat, non-wetting surface is generated by photocuring a film of PFPE-DMAonto a glass slide, according to the procedure outlined for generating apatterned PFPE-DMA mold. 5 μL of the PEG-diacrylate/photoinitiatorsolution is pressed between the PFPE-DMA mold and the flat PFPE-DMAsurface, and pressure is applied to squeeze out excess PEG-diacrylatemonomer. The PFPE-DMA mold is then removed from the flat PFPE-DMAsurface and pressed against a clean glass microscope slide andphotocured using UV radiation (λ=365 nm) for 10 minutes while under anitrogen purge. Particles are observed after cooling to room temperatureand separation of the PFPE mold and the glass microscope slide, usingscanning electron microscopy (SEM) (see FIG. 31).

3.17. Encapsulation of Viruses in PEG-Diacrylate Nanoparticles.

A patterned perfluoropolyether (PFPE) mold is generated by pouringPFPE-dimethacrylate (PFPE-DMA) containing 1-hydroxycyclohexyl phenylketone over a silicon substrate patterned with 200-nm trapezoidal shapes(see FIG. 13). A poly(dimethylsiloxane) mold is used to confine theliquid PFPE-DMA to the desired area. The apparatus is then subjected toUV light (λ=365 nm) for 10 minutes while under a nitrogen purge. Thefully cured PFPE-DMA mold is then released from the silicon master.Separately, a poly(ethylene glycol) (PEG) diacrylate (n=9) is blendedwith 1 wt % of a photoinitiator, 1-hydroxycyclohexyl phenyl ketone.Fluorescently-labeled or unlabeled Adenovirus or Adeno-Associated Virussuspensions are added to this PEG-diacrylate monomer solution and mixedthoroughly. Flat, uniform, non-wetting surfaces are generated bytreating a silicon wafer cleaned with “piranha” solution (1:1concentrated sulfuric acid: 30% hydrogen peroxide (aq) solution) withtrichloro(1H, 1H, 2H, 2H-perfluorooctyl) silane via vapor deposition ina desiccator for 20 minutes. Following this, 50 μL of the PEGdiacrylate/virus solution is then placed on the treated silicon waferand the patterned PFPE mold placed on top of it. The substrate is thenplaced in a molding apparatus and a small pressure is applied to pushout excess PEG-diacrylate solution. The entire apparatus is thensubjected to UV light (λ=365 nm) for ten minutes while under a nitrogenpurge. Virus-containing particles are observed after separation of thePFPE mold and the treated silicon wafer using transmission electronmicroscopy or, in the case of fluorescently-labeled viruses, confocalfluorescence microscopy.

3.18 Encapsulation of Proteins in PEG-Diacrylate Nanoparticles.

A patterned perfluoropolyether (PFPE) mold is generated by pouringPFPE-dimethacrylate (PFPE-DMA) containing 1-hydroxycyclohexyl phenylketone over a silicon substrate patterned with 200-nm trapezoidal shapes(see FIG. 13). A poly(dimethylsiloxane) mold is used to confine theliquid PFPE-DMA to the desired area. The apparatus is then subjected toUV light (λ=365 nm) for 10 minutes while under a nitrogen purge. Thefully cured PFPE-DMA mold is then released from the silicon master.Separately, a poly(ethylene glycol) (PEG) diacrylate (n=9) is blendedwith 1 wt % of a photoinitiator, 1-hydroxycyclohexyl phenyl ketone.Fluorescently-labeled or unlabeled protein solutions are added to thisPEG-diacrylate monomer solution and mixed thoroughly. Flat, uniform,non-wetting surfaces are generated by treating a silicon wafer cleanedwith “piranha” solution (1:1 concentrated sulfuric acid: 30% hydrogenperoxide (aq) solution) with trichloro(1H, 1H, 2H, 2H-perfluorooctyl)silane via vapor deposition in a desiccator for 20 minutes. Followingthis, 50 μL of the PEG diacrylate/virus solution is then placed on thetreated silicon wafer and the patterned PFPE mold placed on top of it.The substrate is then placed in a molding apparatus and a small pressureis applied to push out excess PEG-diacrylate solution. The entireapparatus is then subjected to UV light (λ=365 nm) for ten minutes whileunder a nitrogen purge. Protein-containing particles are observed afterseparation of the PFPE mold and the treated silicon wafer usingtraditional assay methods or, in the case of fluorescently-labeledproteins, confocal fluorescence microscopy.

3.19 Fabrication of 200-nm Titania Particles

A patterned perfluoropolyether (PFPE) mold can be generated by pouring aPFPE-dimethacrylate (PFPE-DMA) containing 1-hydroxycyclohexyl phenylketone over a silicon substrate patterned with 200-nm trapezoidalshapes, such as shown in FIG. 13. A poly(dimethylsiloxane) mold can beused to confine the liquid PFPE-DMA to the desired area. The apparatuscan then be subjected to UV light (λ=365 nm) for 10 minutes while undera nitrogen purge. The fully cured PFPE-DMA mold is then released fromthe silicon master. Separately, 1 g of Pluronic P123 is dissolved in 12g of absolute ethanol. This solution was added to a solution of 2.7 mLof concentrated hydrochloric acid and 3.88 mL titanium (IV) ethoxide.Flat, uniform, non-wetting surfaces can be generated by treating asilicon wafer cleaned with “piranha” solution (1:1 concentrated sulfuricacid: 30% hydrogen peroxide (aq) solution) with trichloro(1H, 1H, 2H,2H-perfluorooctyl) silane via vapor deposition in a desiccator for 20minutes. Following this, 50 μL of the sol-gel solution can then beplaced on the treated silicon wafer and the patterned PFPE mold placedon top of it. The substrate is then placed in a molding apparatus and asmall pressure is applied to push out excess sol-gel precursor. Theentire apparatus is then set aside until the sol-gel precursor hassolidified. After solidification of the sol-gel precursor, the siliconwafer can be removed from the patterned PFPE and particles will bepresent.

3.20 Fabrication of 200-nm Silica Particles

A patterned perfluoropolyether (PFPE) mold can be generated by pouring aPFPE-dimethacrylate (PFPE-DMA) containing 1-hydroxycyclohexyl phenylketone over a silicon substrate patterned with 200-nm trapezoidalshapes, such as shown in FIG. 13. A poly(dimethylsiloxane) mold can thenbe used to confine the liquid PFPE-DMA to the desired area. Theapparatus can then be subjected to UV light (λ=365 nm) for 10 minuteswhile under a nitrogen purge. The fully cured PFPE-DMA mold is thenreleased from the silicon master. Separately, 2 g of Pluronic P123 isdissolved in 30 g of water and 120 g of 2 M HCl is added while stirringat 35° C. To this solution, add 8.50 g of TEOS with stirring at 35° C.for 20 h. Flat, uniform, non-wetting surfaces can then be generated bytreating a silicon wafer cleaned with “piranha” solution (1:1concentrated sulfuric acid: 30% hydrogen peroxide (aq) solution) withtrichloro(1H, 1H, 2H, 2H-perfluorooctyl) silane via vapor deposition ina desiccator for 20 minutes. Following this, 50 μL of the sol-gelsolution is then placed on the treated silicon wafer and the patternedPFPE mold placed on top of it. The substrate is then placed in a moldingapparatus and a small pressure is applied to push out excess sol-gelprecursor. The entire apparatus is then set aside until the sol-gelprecursor has solidified. Particles should be observed after separationof the PFPE mold and the treated silicon wafer using scanning electronmicroscopy (SEM).

3.21 Fabrication of 200-nm Europium-Doped Titania Particles

A patterned perfluoropolyether (PFPE) mold is generated by pouring aPFPE-dimethacrylate (PFPE-DMA) containing 1-hydroxycyclohexyl phenylketone over a silicon substrate patterned with 200-nm trapezoidal shapes(see FIG. 13). A poly(dimethylsiloxane) mold is used to confine theliquid PFPE-DMA to the desired area. The apparatus is then subjected toUV light (λ=365 nm) for 10 minutes while under a nitrogen purge. Thefully cured PFPE-DMA mold is then released from the silicon master.Separately, 1 g of Pluronic P123 and 0.51 g of EuCl₃.6H₂O are dissolvedin 12 g of absolute ethanol. This solution is added to a solution of 2.7mL of concentrated hydrochloric acid and 3.88 mL titanium (IV) ethoxide.Flat, uniform, non-wetting surfaces are generated by treating a siliconwafer cleaned with “piranha” solution (1:1 concentrated sulfuric acid:30% hydrogen peroxide (aq) solution) with trichloro(1H, 1H, 2H,2H-perfluorooctyl) silane via vapor deposition in a desiccator for 20minutes. Following this, 50 μL of the sol-gel solution is then placed onthe treated silicon wafer and the patterned PFPE mold placed on top ofit. The substrate is then placed in a molding apparatus and a smallpressure is applied to push out excess sol-gel precursor. The entireapparatus is then set aside until the sol-gel precursor has solidified.Next, after the sol-gel precursor has solidified, the PFPE mold and thetreated silicon wafer are separated and particles should be observedusing scanning electron microscopy (SEM).

3.22 Encapsulation of CdSe Nanoparticles Inside 200-nm PEG Particles

A patterned perfluoropolyether (PFPE) mold is generated by pouring aPFPE-dimethacrylate (PFPE-DMA) containing 1-hydroxycyclohexyl phenylketone over a silicon substrate patterned with 200-nm trapezoidal shapes(see FIG. 13). A poly(dimethylsiloxane) mold is used to confine theliquid PFPE-DMA to the desired area. The apparatus is then subjected toUV light (λ=365 nm) for 10 minutes while under a nitrogen purge. Thefully cured PFPE-DMA mold is then released from the silicon master.Flat, uniform, non-wetting surfaces are generated by treating a siliconwafer cleaned with “piranha” solution (1:1 concentrated sulfuric acid:30% hydrogen peroxide (aq) solution) with trichloro(1H, 1H, 2H,2H-perfluorooctyl) silane via vapor deposition in a desiccator for 20minutes. Separately, 0.5 g of sodium citrate and 2 mL of 0.04 M cadmiumperchlorate are dissolved in 45 mL of water, and the pH is adjusted toof the solution to 9 with 0.1 M NaOH. The solution is bubbled withnitrogen for 15 minutes. 2 mL of 1 M N,N-dimethylselenourea is added tothe solution and heated in a microwave oven for 60 seconds. 50 μL ofthis solution is added to 50 μL of a PEG diacrylate solution in amicrotube. This microtube is vortexed for ten seconds. 50 μL of this PEGdiacrylate/CdSe particle solution is placed on the treated silicon waferand the patterned PFPE mold placed on top of it. The substrate is thenplaced in a molding apparatus and a small pressure is applied to pushout excess PEG-diacrylate solution. The entire apparatus is thensubjected to UV light (λ=365 nm) for ten minutes while under a nitrogenpurge. PEG-diacrylate particles with encapsulated CdSe nanoparticleswill be observed after separation of the PFPE mold and the treatedsilicon wafer using TEM or fluorescence microscopy.

3.23 Synthetic Replication of Adenovirus Particles Using Non-WettingImprint Lithography

A template, or “master,” for perfluoropolyether-dimethacrylate(PFPE-DMA) mold fabrication is generated by dispersing adenovirusparticles on a silicon wafer. This master can be used to template apatterned mold by pouring PFPE-DMA containing 1-hydroxycyclohexyl phenylketone over the patterned area of the master. A poly(dimethylsiloxane)mold is used to confine the liquid PFPE-DMA to the desired area. Theapparatus is then subjected to UV light (λ=365 nm) for 10 minutes whileunder a nitrogen purge. The fully cured PFPE-DMA mold is then releasedfrom the master. Separately, TMPTA is blended with 1 wt % of aphotoinitiator, 1-hydroxycyclohexyl phenyl ketone. Flat, uniform,non-wetting surfaces are generated by treating a silicon wafer cleanedwith “piranha” solution (1:1 concentrated sulfuric acid: 30% hydrogenperoxide (aq) solution) with trichloro(1H, 1H, 2H, 2H-perfluorooctyl)silane via vapor deposition in a desiccator for 20 minutes. Followingthis, 50 μL of TMPTA is then placed on the treated silicon wafer and thepatterned PFPE mold placed on top of it. The substrate is then placed ina molding apparatus and a small pressure is applied to push out excessTMPTA. The entire apparatus is then subjected to UV light (λ=365 nm) forten minutes while under a nitrogen purge. Synthetic virus replicates areobserved after separation of the PFPE mold and the treated silicon waferusing scanning electron microscopy (SEM) or transmission electronmicroscopy (TEM).

3.24 Synthetic Replication Of Earthworm Hemoglobin Protein UsingNon-Wetting Imprint Lithography

A template, or “master,” for perfluoropolyether-dimethacrylate(PFPE-DMA) mold fabrication is generated by dispersing earthwormhemoglobin protein on a silicon wafer. This master can be used totemplate a patterned mold by pouring PFPE-DMA containing1-hydroxycyclohexyl phenyl ketone over the patterned area of the master.A poly(dimethylsiloxane) mold is used to confine the liquid PFPE-DMA tothe desired area. The apparatus is then subjected to UV light (λ=365 nm)for 10 minutes while under a nitrogen purge. The fully cured PFPE-DMAmold is then released from the master. Separately, TMPTA is blended with1 wt % of a photoinitiator, 1-hydroxycyclohexyl phenyl ketone. Flat,uniform, non-wetting surfaces are generated by treating a silicon wafercleaned with “piranha” solution (1:1 concentrated sulfuric acid: 30%hydrogen peroxide (aq) solution) with trichloro(1H, 1H, 2H,2H-perfluorooctyl) silane via vapor deposition in a desiccator for 20minutes. Following this, 50 μL of TMPTA is then placed on the treatedsilicon wafer and the patterned PFPE mold placed on top of it. Thesubstrate is then placed in a molding apparatus and a small pressure isapplied to push out excess TMPTA. The entire apparatus is then subjectedto UV light (λ=365 nm) for ten minutes while under a nitrogen purge.Synthetic protein replicates are observed after separation of the PFPEmold and the treated silicon wafer using scanning electron microscopy(SEM) or transmission electron microscopy (TEM).

3.25 Combinatorial Engineering of 100-nm Nanoparticle Therapeutics

A patterned perfluoropolyether (PFPE) mold is generated by pouring aPFPE-dimethacrylate (PFPE-DMA) containing 1-hydroxycyclohexyl phenylketone over a silicon substrate patterned with 100-nm cubic shapes. Apoly(dimethylsiloxane) mold is used to confine the liquid PFPE-DMA tothe desired area. The apparatus is then subjected to UV light (λ=365 nm)for 10 minutes while under a nitrogen purge. The fully cured PFPE-DMAmold is then released from the silicon master. Separately, apoly(ethylene glycol) (PEG) diacrylate (n=9) is blended with 1 wt % of aphotoinitiator, 1-hydroxycyclohexyl phenyl ketone. Other therapeuticagents (i.e., small molecule drugs, proteins, polysaccharides, DNA,etc.), tissue targeting agents (cell penetrating peptides and ligands,hormones, antibodies, etc.), therapeutic release/transfection agents(other controlled-release monomer formulations, cationic lipids, etc.),and miscibility enhancing agents (cosolvents, charged monomers, etc.)are added to the polymer precursor solution in a combinatorial manner.Flat, uniform, non-wetting surfaces are generated by treating a siliconwafer cleaned with “piranha” solution (1:1 concentrated sulfuricacid:30% hydrogen peroxide (aq) solution) with trichloro(1H, 1H, 2H,2H-perfluorooctyl) silane via vapor deposition in a desiccator for 20minutes. Following this, 50 μL of the combinatorially-generated particleprecursor solution is then placed on the treated silicon wafer and thepatterned PFPE mold placed on top of it. The substrate is then placed ina molding apparatus and a small pressure is applied to push out excesssolution. The entire apparatus is then subjected to UV light (λ=365 nm)for ten minutes while under a nitrogen purge. The PFPE-DMA mold is thenseparated from the treated wafer, particles can be harvested, and thetherapeutic efficacy of each combinatorially generated nanoparticle isestablished. By repeating this methodology with different particleformulations, many combinations of therapeutic agents, tissue targetingagents, release agents, and other important compounds can be rapidlyscreened to determine the optimal combination for a desired therapeuticapplication.

3.26 Fabrication of a Shape-Specific PEG Membrane

A patterned perfluoropolyether (PFPE) mold is generated by pouring aPFPE-dimethacrylate (PFPE-DMA) containing 1-hydroxycyclohexyl phenylketone over a silicon substrate patterned with 3-μm cylindrical holesthat are 5 μm deep. A poly(dimethylsiloxane) mold is used to confine theliquid PFPE-DMA to the desired area. The apparatus is then subjected toUV light (λ=365 nm) for 10 minutes while under a nitrogen purge. Thefully cured PFPE-DMA mold is then released from the silicon master.Separately, a poly(ethylene glycol) (PEG) diacrylate (n=9) is blendedwith 1 wt % of a photoinitiator, 1-hydroxycyclohexyl phenyl ketone.Flat, uniform, non-wetting surfaces are generated by treating a siliconwafer cleaned with “piranha” solution (1:1 concentrated sulfuricacid:30% hydrogen peroxide (aq) solution) with trichloro(1H, 1H, 2H,2H-perfluorooctyl) silane via vapor deposition in a desiccator for 20minutes. Following this, 50 μL of PEG diacrylate is then placed on thetreated silicon wafer and the patterned PFPE mold placed on top of it.The substrate is then placed in a molding apparatus and a small pressureis applied to push out excess PEG-diacrylate. The entire apparatus isthen subjected to UV light (λ=365 nm) for ten minutes while under anitrogen purge. An interconnected membrane will be observed afterseparation of the PFPE mold and the treated silicon wafer using scanningelectron microscopy (SEM). The membrane will release from the surface bysoaking in water and allowing it to lift off the surface.

3.27 Harvesting of PEG Particles by Ice Formation

A patterned perfluoropolyether (PFPE) mold is generated by pouring aPFPE-dimethacrylate (PFPE-DMA) containing 1-hydroxycyclohexyl phenylketone over a silicon substrate patterned with 5-μm cylinder shapes. Thesubstrate is then subjected to a nitrogen purge for 10 minutes, then UVlight (λ=365 nm) is applied for 10 minutes while under a nitrogen purge.The fully cured PFPE-DMA mold is then released from the silicon master.Separately, a poly(ethylene glycol) (PEG) diacrylate (n=9) is blendedwith 1 wt % of a photoinitiator, 1-hydroxycyclohexyl phenyl ketone.Flat, uniform, non-wetting surfaces are generated by coating a glassslide with PFPE-DMA containing 1-hydroxycyclohexyl phenyl ketone. Theslide is then subjected to a nitrogen purge for 10 minutes, then UVlight (λ=365 nm) is applied for 10 minutes while under a nitrogen purge.The flat, fully cured PFPE-DMA substrate is released from the slide.Following this, 0.1 mL of PEG diacrylate is then placed on the flatPFPE-DMA substrate and the patterned PFPE mold placed on top of it. Thesubstrate is then placed in a molding apparatus and a small pressure isapplied to push out excess PEG-diacrylate. The entire apparatus is thenpurged with nitrogen for 10 minutes, then subjected to UV light (λ=365nm) for 10 minutes while under a nitrogen purge. PEG particles areobserved after separation of the PFPE-DMA mold and substrate usingoptical microscopy. Water is applied to the surface of the substrate andmold containing particles. A gasket is used to confine the water to thedesired location. The apparatus is then placed in the freezer at atemperature of −10° C. for 30 minutes. The ice containing PEG particlesis peeled off the PFPE-DMA mold and substrate and allowed to melt,yielding an aqueous solution containing PEG particles.

3.28 Harvesting of PEG Particles with Vinyl Pyrrolidone

A patterned perfluoropolyether (PFPE) mold is generated by pouring aPFPE-dimethacrylate (PFPE-DMA) containing 1-hydroxycyclohexyl phenylketone over a silicon substrate patterned with 5-μm cylinder shapes. Thesubstrate is then subjected to a nitrogen purge for 10 minutes, and thenUV light (λ=365 nm) is applied for 10 minutes while under a nitrogenpurge. The fully cured PFPE-DMA mold is then released from the siliconmaster. Separately, a poly(ethylene glycol) (PEG) diacrylate (n=9) isblended with 1 wt % of a photoinitiator, 1-hydroxycyclohexyl phenylketone. Flat, uniform, non-wetting surfaces are generated by coating aglass slide with PFPE-DMA containing 1-hydroxycyclohexyl phenyl ketone.The slide is then subjected to a nitrogen purge for 10 minutes, then UVlight (λ=365 nm) is applied for 10 minutes while under a nitrogen purge.The flat, fully cured PFPE-DMA substrate is released from the slide.Following this, 0.1 mL of PEG diacrylate is then placed on the flatPFPE-DMA substrate and the patterned PFPE mold placed on top of it. Thesubstrate is then placed in a molding apparatus and a small pressure isapplied to push out excess PEG-diacrylate. The entire apparatus is thenpurged with nitrogen for 10 minutes, then subjected to UV light (λ=365nm) for 10 minutes while under a nitrogen purge. PEG particles areobserved after separation of the PFPE-DMA mold and substrate usingoptical microscopy. In some embodiments, the material includes anadhesive or sticky surface. In some embodiments, the material includescarbohydrates, epoxies, waxes, polyvinyl alcohol, polyvinyl pyrrolidone,polybutyl acrylate, polycyano acrylates, polymethyl methacrylate. Insome embodiments, the harvesting or collecting of the particles includescooling water to form ice (e.g., in contact with the particles) drop ofn-vinyl-2-pyrrolidone containing 5% photoinitiator, 1-hydroxycyclohexylphenyl ketone, is placed on a clean glass slide. The PFPE-DMA moldcontaining particles is placed patterned side down on then-vinyl-2-pyrrolidone drop. The slide is subjected to a nitrogen purgefor 5 minutes, then UV light (λ=365 nm) is applied for 5 minutes whileunder a nitrogen purge. The slide is removed, and the mold is peeledaway from the polyvinyl pyrrolidone and particles. Particles on thepolyvinyl pyrrolidone were observed with optical microscopy. Thepolyvinyl pyrrolidone film containing particles was dissolved in water.Dialysis was used to remove the polyvinyl pyrrolidone, leaving anaqueous solution containing 5 μm PEG particles.

3.29 Harvesting of PEG Particles with Polyvinyl Alcohol

A patterned perfluoropolyether (PFPE) mold is generated by pouring aPFPE-dimethacrylate (PFPE-DMA) containing 1-hydroxycyclohexyl phenylketone over a silicon substrate patterned with 5-μm cylinder shapes. Thesubstrate is then subjected to a nitrogen purge for 10 minutes, then UVlight (λ=365 nm) is applied for 10 minutes while under a nitrogen purge.The fully cured PFPE-DMA mold is then released from the silicon master.Separately, a poly(ethylene glycol) (PEG) diacrylate (n=9) is blendedwith 1 wt % of a photoinitiator, 1-hydroxycyclohexyl phenyl ketone.Flat, uniform, non-wetting surfaces are generated by coating a glassslide with PFPE-DMA containing 1-hydroxycyclohexyl phenyl ketone. Theslide is then subjected to a nitrogen purge for 10 minutes, then UVlight (λ=365 nm) is applied for 10 minutes while under a nitrogen purge.The flat, fully cured PFPE-DMA substrate is released from the slide.Following this, 0.1 mL of PEG diacrylate is then placed on the flatPFPE-DMA substrate and the patterned PFPE mold placed on top of it. Thesubstrate is then placed in a molding apparatus and a small pressure isapplied to push out excess PEG-diacrylate. The entire apparatus is thenpurged with nitrogen for 10 minutes, then subjected to UV light (λ=365nm) for 10 minutes while under a nitrogen purge. PEG particles areobserved after separation of the PFPE-DMA mold and substrate usingoptical microscopy. Separately, a solution of 5 weight percent polyvinylalcohol (PVOH) in ethanol (EtOH) is prepared. The solution is spincoated on a glass slide and allowed to dry. The PFPE-DMA mold containingparticles is placed patterned side down on the glass slide and pressureis applied. The mold is then peeled away from the PVOH and particles.Particles on the PVOH were observed with optical microscopy. The PVOHfilm containing particles was dissolved in water. Dialysis was used toremove the PVOH, leaving an aqueous solution containing 5 μm PEGparticles.

3.30 Fabrication of 200 Nm Phosphatidylcholine Particles

A patterned perfluoropolyether (PFPE) mold is generated by pouring aPFPE-dimethacrylate (PFPE-DMA) containing 1-hydroxycyclohexyl phenylketone over a silicon substrate patterned with 200-nm trapezoidal shapes(see FIG. 13). A poly(dimethylsiloxane) mold is used to confine theliquid PFPE-DMA to the desired area. The apparatus is then subjected toa nitrogen purge for 10 minutes followed by UV light (λ=365 nm) for 10minutes while under a nitrogen purge. The fully cured PFPE-DMA mold isthen released from the silicon master. Separately, flat, uniform,non-wetting surfaces are generated by treating a silicon wafer cleanedwith “piranha” solution (1:1 concentrated sulfuric acid: 30% hydrogenperoxide (aq) solution) with trichloro(1H, 1H, 2H, 2H-perfluorooctyl)silane via vapor deposition in a desiccator for 20 minutes. Followingthis, 20 mg of the phosphatidylcholine was placed on the treated siliconwafer and heated to 60 degrees C. The substrate is then placed in amolding apparatus and a small pressure is applied to push out excessphosphatidylcholine. The entire apparatus is then set aside until thephosphatidylcholine has solidified. Particles are observed afterseparation of the PFPE mold and the treated silicon wafer using scanningelectron microscopy (SEM).

3.31 Functionalizing PEG Particles with FITC

Poly(ethylene glycol) (PEG) particles with 5 weight percent aminoethylmethacrylate were created. Particles are observed in the PFPE mold afterseparation of the PFPE mold and the PFPE substrate using opticalmicroscopy. Separately, a solution containing 10 weight percentfluorescein isothiocyanate (FITC) in dimethylsulfoxide (DMSO) wascreated. Following this, the mold containing the particles was exposedto the FITC solution for one hour. Excess FITC was rinsed off the moldsurface with DMSO followed by deionized (DI) water. The tagged particleswere observed with fluorescence microscopy, with an excitationwavelength of 492 nm and an emission wavelength of 529 nm.

3.32 Encapsulation of Doxorubicin Inside 500 nm Conical PEG Particles

A patterned perfluoropolyether (PFPE) mold was generated by pouring aPFPE-dimethacrylate (PFPE-DMA) containing 1-hydroxycyclohexyl phenylketone over a silicon substrate patterned with 500-nm conical shapes(see FIG. 15). A poly(dimethylsiloxane) mold was used to confine theliquid PFPE-DMA to the desired area. The apparatus was then subjected toUV light (λ=365 nm) for 10 minutes while under a nitrogen purge. Thefully cured PFPE-DMA mold was then released from the silicon master.Flat, uniform, non-wetting surfaces were generated by treating a siliconwafer cleaned with “piranha” solution (1:1 concentrated sulfuric acid:30% hydrogen peroxide (aq) solution) with trichloro(1H, 1H, 2H,2H-perfluorooctyl) silane via vapor deposition in a desiccator for 20minutes. Separately, a solution of 1 wt % doxorubicin in PEG diacrylatewas formulated with 1 wt % photoinitiator. Following this, 50 μL of thisPEG diacrylate/doxorubicin solution was then placed on the treatedsilicon wafer and the patterned PFPE mold placed on top of it. Thesubstrate was then placed in a molding apparatus and a small pressurewas applied to push out excess PEG-diacrylate/doxorubicin solution. Thesmall pressure in this example was at least about 100 N/cm². The entireapparatus was then subjected to UV light (λ=365 nm) for ten minuteswhile under a nitrogen purge. Doxorubicin-containing PEG-diacrylateparticles were observed after separation of the PFPE mold and thetreated silicon wafer using fluorescent microscopy (see FIG. 32).

3.33 Encapsulation of Avidin (66 kDa) in 160 nm PEG Particles

A patterned perfluoropolyether (PFPE) mold was generated by pouring aPFPE-dimethacrylate (PFPE-DMA) containing 1-hydroxycyclohexyl phenylketone over a silicon substrate patterned with 160-nm cylindrical shapes(see FIG. 33). A poly(dimethylsiloxane) mold was used to confine theliquid PFPE-DMA to the desired area. The apparatus was then subjected toUV light (λ=365 nm) for 10 minutes white under a nitrogen purge. Thefully cured PFPE-DMA mold was then released from the silicon master.Flat, uniform, non-wetting surfaces are generated by treating a siliconwafer cleaned with “piranha” solution (1:1 concentrated sulfuric acid:30% hydrogen peroxide (aq) solution) with trichloro(1H, 1H, 2H,2H-perfluorooctyl) silane via vapor deposition in a desiccator for 20minutes. Separately, a solution of 1 wt % avidin in 30:70 PEGmonomethacrylate:PEG diacrylate was formulated with 1 wt %photoinitiator. Following this, 50 μL of this PEG/avidin solution wasthen placed on the treated silicon wafer and the patterned PFPE moldplaced on top of it. The substrate was then placed in a moldingapparatus and a small pressure is applied to push out excessPEG-diacrylate/avidin solution. The small pressure in this example wasat least about 100 N/cm². The entire apparatus was then subjected to UVlight (λ=365 nm) for ten minutes while under a nitrogen purge.Avidin-containing PEG particles were observed after separation of thePFPE mold and the treated silicon wafer using fluorescent microscopy.

3.34 Encapsulation of 2-fluoro-2-deoxy-d-glucose in 80 nm PEG Particles

A patterned perfluoropolyether (PFPE) mold is generated by pouring aPFPE-dimethacrylate (PFPE-DMA) containing 1-hydroxycyclohexyl phenylketone over a 6 inch silicon substrate patterned with 80-nm cylindricalshapes. The substrate is then subjected to UV light (λ=365 nm) for 10minutes while under a nitrogen purge. The fully cured PFPE-DMA mold isthen released from the silicon master. Flat, uniform, non-wettingsurfaces are generated by treating a silicon wafer cleaned with“piranha” solution (1:1 concentrated sulfuric acid: 30% hydrogenperoxide (aq) solution) with trichloro(1H, 1H, 2H, 2H-perfluorooctyl)silane via vapor deposition in a desiccator for 20 minutes. Separately,a solution of 0.5 wt % 2-fluoro-2-deoxy-d-glucose (FDG) in 30:70 PEGmonomethacrylate:PEG diacrylate is formulated with 1 wt %photoinitiator. Following this, 200 μL of this PEG/FDG solution is thenplaced on the treated silicon wafer and the patterned PFPE mold placedon top of it. The substrate is then placed in a molding apparatus and asmall pressure is applied to push out excess PEG/FDG solution. The smallpressure should be at least about 100 N/cm². The entire apparatus isthen subjected to UV light (λ=365′ nm) for ten minutes while under anitrogen purge. FDG-containing PEG particles will be observed afterseparation of the PFPE mold and the treated silicon wafer using scanningelectron microscopy.

3.35 Encapsulated DNA in 200 nm×200 nm×1 μm Bar-Shaped Poly(Lactic Acid)Particles

A patterned perfluoropolyether (PFPE) mold is generated by pouring aPFPE-dimethacrylate (PFPE-DMA) containing 1-hydroxycyclohexyl phenylketone over a silicon substrate patterned with 200 nm×200 nm×1 μm barshapes. The substrate is then subjected to UV light (λ=365 nm) for 10minutes while under a nitrogen purge. The fully cured PFPE-DMA mold isthen released from the silicon master. Flat, uniform, non-wettingsurfaces are generated by treating a silicon wafer cleaned with“piranha” solution (1:1 concentrated sulfuric acid: 30% hydrogenperoxide (aq) solution) with trichloro(1H, 1H, 2H, 2H-perfluorooctyl)silane via vapor deposition in a desiccator for 20 minutes. Separately,a solution of 0.01 wt % 24 base pair DNA and 5 wt % poly(lactic acid) inethanol is formulated. 200 μL of this ethanol solution is then placed onthe treated silicon wafer and the patterned PFPE mold placed on top ofit. The substrate is then placed in a molding apparatus and a smallpressure is applied to push out excess PEG/FDG solution. The smallpressure should be at least about 100 N/cm². The entire apparatus isthen placed under vacuum for 2 hours. DNA-containing poly(lactic acid)particles will be observed after separation of the PFPE mold and thetreated silicon wafer using optical microscopy.

3.36 100 nm Paclitaxel Particles

A patterned perfluoropolyether (PFPE) mold is generated by pouring aPFPE-dimethacrylate (PFPE-DMA) containing 1-hydroxycyclohexyl phenylketone over a silicon substrate patterned with 500-nm conical shapes(see FIG. 15). A poly(dimethylsiloxane) mold is used to confine theliquid PFPE-DMA to the desired area. The apparatus is then subjected toUV light (λ=365 nm) for 10 minutes while under a nitrogen purge. Thefully cured PFPE-DMA mold is then released from the silicon master.Flat, uniform, non-wetting surfaces are generated by treating a siliconwafer cleaned with “piranha” solution (1:1 concentrated sulfuric acid:30% hydrogen peroxide (aq) solution) with trichloro(1H, 1H, 2H,2H-perfluorooctyl) silane via vapor deposition in a desiccator for 20minutes. Separately, a solution of 5 wt % paclitaxel in ethanol wasformulated. Following this, 100 μL of this paclitaxel solution is thenplaced on the treated silicon wafer and the patterned PFPE mold placedon top of it. The substrate is then placed in a molding apparatus and asmall pressure is applied to push out excess solution. The pressureapplied was at least about 100 N/cm². The entire apparatus is thenplaced under vacuum for 2 hours. Separation of the mold and surfaceyielded approximately 100 nm spherical paclitaxel particles, which wereobserved with scanning electron microscopy.

3.37 Triangular Particles Functionalized on One Side

A patterned perfluoropolyether (PFPE) mold is generated by pouring aPFPE-dimethacrylate (PFPE-DMA) containing 1-hydroxycyclohexyl phenylketone over a 6 inch silicon substrate patterned with 0.6 μm×0.8 μm×1 μmright triangles. The substrate is then subjected to UV light (λ=365 nm)for 10 minutes while under a nitrogen purge. The fully cured PFPE-DMAmold is then released from the silicon master. Flat, uniform,non-wetting surfaces are generated by treating a silicon wafer cleanedwith “piranha” solution (1:1 concentrated sulfuric acid: 30% hydrogenperoxide (aq) solution) with trichloro(1H, 1H, 2H, 2H-perfluorooctyl)silane via vapor deposition in a desiccator for 20 minutes. Separately,a solution of 5 wt % aminoethyl methacrylate in 30:70 PEGmonomethacrylate:PEG diacrylate is formulated with 1 wt %photoinitiator. Following this, 200 μL of this monomer solution is thenplaced on the treated silicon wafer and the patterned PFPE mold placedon top of it. The substrate is then placed in a molding apparatus and asmall pressure is applied to push out excess solution. The smallpressure should be at least about 100 N/cm². The entire apparatus isthen subjected to UV light (λ=365 nm) for ten minutes while under anitrogen purge. Aminoethyl methacrylate-containing PEG particles areobserved in the mold after separation of the PFPE mold and the treatedsilicon wafer using optical microscopy. Separately, a solutioncontaining 10 weight percent fluorescein isothiocyanate (FITC) indimethylsulfoxide (DMSO) is created. Following this, the mold containingthe particles is exposed to the FITC solution for one hour. Excess FITCis rinsed off the mold surface with DMSO followed by deionized (DI)water. Particles, tagged only on one face, will be observed withfluorescence microscopy, with an excitation wavelength of 492 nm and anemission wavelength of 529 nm.

3.38 Formation of an Imprinted Protein Binding Cavity and an ArtificialProtein.

The desired protein molecules are adsorbed onto a mica substrate tocreate a master template. A mixture of PFPE-dimethacrylate (PFPE-DMA)containing a monomer with a covalently attached disaccharide, and1-hydroxycyclohexyl phenyl ketone as a photoinitiator was poured overthe substrate. The substrate is then subjected to UV light (λ=365 nm)for 10 minutes while under a nitrogen purge. The fully cured PFPE-DMAmold is then released from the mica master, creating polysaccharide-likecavities that exhibit selective recognition for the protein moleculethat was imprinted. The polymeric mold was soaked in NaOH/NaClO solutionto remove the template proteins.

Flat, uniform, non-wetting surfaces are generated by treating a siliconwafer cleaned with “piranha” solution (1:1 concentrated sulfuric acid:30% hydrogen peroxide (aq) solution) with trichloro(1H, 1H, 2H,2H-perfluorooctyl) silane via vapor deposition in a desiccator for 20minutes. Separately, a solution of 25% (w/w) methacrylic acid (MAA), 25%diethyl aminoethylmethacrylate (DEAEM), and 48% PEG diacrylate wasformulated with 2 wt % photoinitiator. Following this, 200 μL of thismonomer solution is then placed on the treated silicon wafer and thepatterned PFPE/disaccharide mold placed on top of it. The substrate isthen placed in a molding apparatus and a small pressure is applied topush out excess solution. The entire apparatus is then subjected to UVlight (λ=365 nm) for ten minutes while under a nitrogen purge. Removalof the mold yields artificial protein molecules which have similar size,shape, and chemical functionality as the original template proteinmolecule.

3.39 Template Filling with “Moving Drop”

A mold (6 inch in diameter) with 5×5×10 micron pattern was placed on aninclined surface that has an angle of 20 degrees to horizon. Then a setof 100 μL drops of 98% PEG-diacrylate and 2% photo initiator solutionwas placed on the surface of the mold at a higher end. Each drop thenwould slide down leaving the trace with filled cavities.

After all the drops reached the lower end the mold was put in UV oven,purged with nitrogen for 15 minutes and then cured for 15 minutes. Theparticles were harvested on glass slide using cyanoacrylate adhesive. Noscum was detected and monodispersity of the particles was confirmedfirst using optical microscope and then scanning electron microscope.

3.40 Template Filling Through Dipping

A mold of size 0.5×3 cm with 3×3×8 micron pattern was dipped into thevial with 98% PEG-diacrylate and 2% photo initiator solution. After 30seconds the mold was withdrawn at a rate of approximately 1 mm persecond.

Then the mold was put into an UV oven, purged with nitrogen for 15minutes, and then cured for 15 minutes. The particles were harvested onthe glass slide using cyanoacrylate adhesive. No scum was detected andmonodispersity of the particles was confirmed using optical microscope.

3.41 Template Filling by Voltage Assist

A voltage of about 3000 volts DC can be applied across a substance to bemolded, such as PEG. The voltage makes the filling process easier as itchanges the contact angle of substance on the patterned template.

3.42 Fabrication of 2 μm Cube-Shaped PEG Particles by Dipping

A patterned perfluoropolyether (PFPE) mold is generated by pouring aPFPE-dimethacrylate (PFPE-DMA) containing 1-hydroxycyclohexyl phenylketone over a silicon substrate patterned with 2-μm×2-μm×1-μm cubes. Theapparatus is then subjected to UV light (λ=365 nm) for 10 minutes whileunder a nitrogen purge. The fully cured PFPE-DMA mold is then releasedfrom the silicon master. Separately, a poly(ethylene glycol) (PEG)diacrylate (n=9) is blended with 1 wt % of a photoinitiator,1-hydroxycyclohexyl phenyl ketone. Fluorescently-labeled methacrylate isadded to this PEG-diacrylate monomer solution and mixed thoroughly. Themold is dipped into this solution and withdrawn slowly. The mold issubjected to UV light for 10 minutes under nitrogen purge. The particlesare harvested by placing cyanoacrylate onto a glass slide, placing themold in contact with the cyanoacrylate, and allowing the cyanoacrylateto cure. The mold is removed from the cured film, leaving the particlesentrapped in the film. The cyanoacrylate is dissolved away usingacetone, and the particles are collected in an acetone solution, andpurified with centrifugation. Particles are observed using scanningelectron microscopy (SEM) after drying (see FIGS. 34A and 34B).

Example 4 Molding of Features for Semiconductor Applications

4.1 Fabrication of 140-nm Lines Separated by 70 nm in TMPTA

A patterned perfluoropolyether (PFPE) mold is generated by pouring aPFPE-dimethacrylate (PFPE-DMA) containing 1-hydroxycyclohexyl phenylketone over a silicon substrate patterned with 140-nm lines separated by70 nm. A poly(dimethylsiloxane) mold is used to confine the liquidPFPE-DMA to the desired area. The apparatus is then subjected to UVlight (λ=365 nm) for 10 minutes while under a nitrogen purge. The fullycured PFPE-DMA mold is then released from the silicon master.Separately, TMPTA is blended with 1 wt % of a photoinitiator,1-hydroxycyclohexyl phenyl ketone. Flat, uniform, surfaces are generatedby treating a silicon wafer cleaned with “piranha” solution (1:1concentrated sulfuric acid:30% hydrogen peroxide (aq) solution) andtreating the wafer with an adhesion promoter, (trimethoxysilyl propylmethacryalte). Following this, 50 μL of TMPTA is then placed on thetreated silicon wafer and the patterned PFPE mold placed on top of it:The substrate is then placed in a molding apparatus and a small pressureis applied to ensure a conformal contact. The entire apparatus is thensubjected to UV light (λ=365 nm) for ten minutes while under a nitrogenpurge. Features are observed after separation of the PFPE mold and thetreated silicon wafer using atomic force microscopy (AFM) (see FIG. 35).

4.2 Molding of a Polystyrene Solution

A patterned perfluoropolyether (PFPE) mold is generated by pouring aPFPE-dimethacrylate (PFPE-DMA) containing 1-hydroxycyclohexyl phenylketone over a silicon substrate patterned with 140-nm lines separated by70 nm. A poly(dimethylsiloxane) mold is used to confine the liquidPFPE-DMA to the desired area. The apparatus is then subjected to UVlight (λ=365 nm) for 10 minutes while under a nitrogen purge. The fullycured PFPE-DMA mold is then released from the silicon master.Separately, polystyrene is dissolved in 1 to 99 wt % of toluene. Flat,uniform, surfaces are generated by treating a silicon wafer cleaned with“piranha” solution (1:1 concentrated sulfuric acid:30% hydrogen peroxide(aq) solution) and treating the wafer with an adhesion promoter.Following this, 50 μL of polystyrene solution is then placed on thetreated silicon wafer and the patterned PFPE mold is placed on top ofit. The substrate is then placed in a molding apparatus and a smallpressure is applied to ensure a conformal contact. The entire apparatusis then subjected to vacuum for a period of time to remove the solvent.Features are observed after separation of the PFPE mold and the treatedsilicon wafer using atomic force microscopy (AFM) and scanning electronmicroscopy (SEM).

4.3 Molding of Isolated Features on Microelectronics-Compatible SurfacesUsing “Double Stamping”

A patterned perfluoropolyether (PFPE) mold is generated by pouring aPFPE-dimethacrylate (PFPE-DMA) containing 1-hydroxycyclohexyl phenylketone over a silicon substrate patterned with 140-nm lines separated by70 nm. A poly(dimethylsiloxane) mold is used to confine the liquidPFPE-DMA to the desired area. The apparatus is then subjected to UVlight (λ=365 nm) for 10 minutes while under a nitrogen purge. The fullycured PFPE-DMA mold is then released from the silicon master.Separately, TMPTA is blended with 1 wt % of a photoinitiator,1-hydroxycyclohexyl phenyl ketone. A flat, non-wetting surface isgenerated by photocuring a film of PFPE-DMA onto a glass slide,according to the procedure outlined for generating a patterned PFPE-DMAmold. 50 μL of the TMPTA/photoinitiator solution is pressed between thePFPE-DMA mold and the flat PFPE-DMA surface, and pressure is applied tosqueeze out excess TMPTA monomer. The PFPE-DMA mold is then removed fromthe flat PFPE-DMA surface and pressed against a clean, flatsilicon/silicon oxide wafer and photocured using UV radiation (λ=365 nm)for 10 minutes while under a nitrogen purge. Isolated, poly(TMPTA)features are observed after separation of the PFPE mold and thesilicon/silicon oxide wafer, using scanning electron microscopy (SEM).

4.4 Fabrication of 200-nm Titania Structures for Microelectronics

A patterned perfluoropolyether (PFPE) mold is generated by pouring aPFPE-dimethacrylate (PFPE-DMA) containing 1-hydroxycyclohexyl phenylketone over a silicon substrate patterned with 140-nm lines separated by70 nm. A poly(dimethylsiloxane) mold is used to confine the liquidPFPE-DMA to the desired area. The apparatus is then subjected to UVlight (λ=365 nm) for 10 minutes while under a nitrogen purge. The fullycured PFPE-DMA mold is then released from the silicon master.Separately, 1 g of Pluronic P123 is dissolved in 12 g of absoluteethanol. This solution was added to a solution of 2.7 mL of concentratedhydrochloric acid and 3.88 mL titanium (IV) ethoxide. Flat, uniform,surfaces are generated by treating a silicon/silicon oxide wafer with“piranha” solution (1:1 concentrated sulfuric acid:30% hydrogen peroxide(aq) solution) and drying. Following this, 50 μL of the sol-gel solutionis then placed on the treated silicon wafer and the patterned PFPE moldplaced on top of it. The substrate is then placed in a molding apparatusand a small pressure is applied to push out excess sol-gel precursor.The entire apparatus is then set aside until the sot-gel precursor hassolidified. Oxide structures will be observed after separation of thePFPE mold and the treated silicon wafer using scanning electronmicroscopy (SEM).

4.5 Fabrication of 200-nm Silica Structures for Microelectronics

A patterned perfluoropolyether (PFPE) mold is generated by pouring aPFPE-dimethacrylate (PFPE-DMA) containing 1-hydroxycyclohexyl phenylketone over a silicon substrate patterned with 140-nm lines separated by70 nm. A poly(dimethylsiloxane) mold is used to confine the liquidPFPE-DMA to the desired area. The apparatus is then subjected to UVlight (λ=365 nm) for 10 minutes while under a nitrogen purge. The fullycured PFPE-DMA mold is then released from the silicon master.Separately, 2 g of Pluronic P123 is dissolved in 30 g of water and 120 gof 2 M HCl is added while stirring at 35° C. To this solution, add 8.50g of TEOS with stirring at 35° C. for 20 h. Flat, uniform, surfaces aregenerated by treating a silicon/silicon oxide wafer with “piranha”solution (1:1 concentrated sulfuric acid:30% hydrogen peroxide (aq)solution) and drying. Following this, 50 μL of the sol-gel solution isthen placed on the treated silicon wafer and the patterned PFPE moldplaced on top of it. The substrate is then placed in a molding apparatusand a small pressure is applied to push out excess sol-gel precursor.The entire apparatus is then set aside until the sol gel precursor hassolidified. Oxide structures will be observed after separation of thePFPE mold and the treated silicon wafer using scanning electronmicroscopy (SEM).

4.6 Fabrication of 200-nm Europium-Doped Titania Structures forMicroelectronics

A patterned perfluoropolyether (PFPE) mold is generated by pouring aPFPE-dimethacrylate (PFPE-DMA) containing 1-hydroxycyclohexyl phenylketone over a silicon substrate patterned with 140-nm lines separated by70 nm. A poly(dimethylsiloxane) mold is used to confine the liquidPFPE-DMA to the desired area. The apparatus is then subjected to UVlight (λ=365 nm) for 10 minutes while under a nitrogen purge. The fullycured PFPE-DMA mold is then released from the silicon master.Separately, 1 g of Pluronic P123 and 0.51 g of EuCl₃.6H₂O are dissolvedin 12 g of absolute ethanol. This solution was added to a solution of2.7 mL of concentrated hydrochloric acid and 3.88 mL titanium (IV)ethoxide. Flat, uniform, surfaces are generated by treating asilicon/silicon oxide wafer with “piranha” solution (1:1 concentratedsulfuric acid:30% hydrogen peroxide (aq) solution) and drying. Followingthis, 50 μL of the sol-gel solution is then placed on the treatedsilicon wafer and the patterned PFPE mold placed on top of it. Thesubstrate is then placed in a molding apparatus and a small pressure isapplied to push out excess sol-gel precursor. The entire apparatus isthen set aside until the sol-gel precursor has solidified. Oxidestructures will be observed after separation of the PFPE mold and thetreated silicon wafer using scanning electron microscopy (SEM).

4.7 Fabrication of Isolated “Scum Free” Features for Microelectronics

A patterned perfluoropolyether (PFPE) mold is generated by pouring aPFPE-dimethacrylate (PFPE-DMA) containing 1-hydroxycyclohexyl phenylketone over a silicon substrate patterned with 140-nm lines separated by70 nm. A poly(dimethylsiloxane) mold is used to confine the liquidPFPE-DMA to the desired area. The apparatus is then subjected to UVlight (λ=365 nm) for 10 minutes while under a nitrogen purge. The fullycured PFPE-DMA mold is then released from the silicon master.Separately, TMPTA is blended with 1 wt % of a photoinitiator,1-hydroxycyclohexyl phenyl ketone. Flat, uniform, non-wetting surfacescapable of adhering to the resist material are generated by treating asilicon wafer cleaned with “piranha” solution (1:1 concentrated sulfuricacid:30% hydrogen peroxide (aq) solution) and treating the wafer with amixture of an adhesion promoter, (trimethoxysilyl propyl methacrylate)and a non-wetting silane agent (1H, 1H, 2H, 2H-perfluorooctyltrimethoxysilane). The mixture can range from 100% of the adhesionpromoter to 100% of the non-wetting silane. Following this, 50 μL ofTMPTA is then placed on the treated silicon wafer and the patterned PFPEmold placed on top of it. The substrate is then placed in a moldingapparatus and a small pressure is applied to ensure a conformal contactand to push out excess TMPTA. The entire apparatus is then subjected toUV light (λ=365 nm) for ten minutes while under a nitrogen purge.Features are observed after separation of the PFPE mold and the treatedsilicon wafer using atomic force microscopy (AFM) and scanning electronmicroscopy (SEM).

Example 5 Molding of Natural and Engineered Templates

5.1. Fabrication of a Perfluoropolyether-Dimethacrylate (PFPE-DMA) Moldfrom a Template Generated Using Electron-Beam Lithography

A template, or “master,” for perfluoropolyether-dimethacrylate(PFPE-DMA) mold fabrication is generated using electron beam lithographyby spin coating a bilayer resist of 200,000 MW PMMA and 900,000 MW PMMAonto a silicon wafer with 500-nm thermal oxide, and exposing this resistlayer to an electron beam that is translating in a pre-programmedpattern. The resist is developed in 3:1 isopropanol:methyl isobutylketone solution to remove exposed regions of the resist. A correspondingmetal pattern is formed on the silicon oxide surface by evaporating 5 nmCr and 15 nm Au onto the resist covered surface and lifting off theresidual PMMA/Cr/Au film in refluxing acetone. This pattern istransferred to the underlying silicon oxide surface by reactive ionetching with CF₄/O₂ plasma and removal of the Cr/Au film in aqua regia(see FIG. 36). This master can be used to template a patterned mold bypouring PFPE-DMA containing 1-hydroxycyclohexyl phenyl ketone over thepatterned area of the master. A poly(dimethylsiloxane) mold is used toconfine the liquid PFPE-DMA to the desired area. The apparatus is thensubjected to UV light (λ=365 nm) for 10 minutes while under a nitrogenpurge. The fully cured PFPE-DMA mold is then released from the master.This mold can be used for the fabrication of particles using non-wettingimprint lithography as specified in Particle Fabrication Examples 3.3and 3.4.

5.2 Fabrication of a Perfluoropolyether-Dimethacrylate (PFPE-DMA Moldfrom a Template Generated Using Photolithography.

A template, or “master,” for perfluoropolyether-dimethacrylate(PFPE-DMA) mold fabrication is generated using photolithography by spincoating a film of SU-8 photoresist onto a silicon wafer. This resist isbaked on a hotplate at 95° C. and exposed through a pre-patternedphotomask. The wafer is baked again at 95° C. and developed using acommercial developer solution to remove unexposed SU-8 resist. Theresulting patterned surface is fully cured at 175° C. This master can beused to template a patterned mold by pouring PFPE-DMA containing1-hydroxycyclohexyl phenyl ketone over the patterned area of the master.A poly(dimethylsiloxane) mold is used to confine the liquid PFPE-DMA tothe desired area. The apparatus is then subjected to UV light (λ=365 nm)for 10 minutes while under a nitrogen purge. The fully cured PFPE-DMAmold is then released from the master, and can be imaged by opticalmicroscopy to reveal the patterned PFPE-DMA mold (see FIGS. 37A and37B).

5.3 Fabrication of a Perfluoropolyether-Dimethacrylate PFPE-DMA) Moldfrom a Template Generated from Dispersed Tobacco Mosaic Virus Particles

A template, or “master,” for perfluoropolyether-dimethacrylate(PFPE-DMA) mold fabrication is generated by dispersing tobacco mosaicvirus (TMV) particles on a silicon wafer (FIG. 38a ). This master can beused to template a patterned mold by pouring PFPE-DMA containing1-hydroxycyclohexyl phenyl ketone over the patterned area of the master.A poly(dimethylsiloxane) mold is used to confine the liquid PFPE-DMA tothe desired area. The apparatus is then subjected to UV light (λ=365 nm)for 10 minutes while under a nitrogen purge. The fully cured PFPE-DMAmold is then released from the master. The morphology of the mold canthen be confirmed using Atomic Force Microscopy (FIG. 38b ).

5.4 Fabrication of a Perfluoropolyether-Dimethacrylate (PFPE-DMA) Moldfrom a Template Generated from Block-Copolymer Micelles

A template, or “master,” for perfluoropolyether-dimethacrylate(PFPE-DMA) mold fabrication is generated by dispersingpolystyrene-polyisoprene block copolymer micelles on a freshly-cleavedmica surface. This master can be used to template a patterned mold bypouring PFPE-DMA containing 1-hydroxycyclohexyl phenyl ketone over thepatterned area of the master. A poly(dimethylsiloxane) mold is used toconfine the liquid PFPE-DMA to the desired area. The apparatus is thensubjected to UV light (λ=365 nm) for 10 minutes while under a nitrogenpurge. The fully cured PFPE-DMA mold is then released from the master.The morphology of the mold can then be confirmed using Atomic ForceMicroscopy (see FIG. 39).

5.5 Fabrication of a Perfluoropolyether-Dimethacrylate (PFPE-DMA) Moldfrom a Template Generated from Brush Polymers.

A template, or “master,” for perfluoropolyether-dimethacrylate(PFPE-DMA) mold fabrication is generated by dispersing poly(butylacrylate) brush polymers on a freshly-cleaved mica surface. This mastercan be used to template a patterned mold by pouring PFPE-DMA containing1-hydroxycyclohexyl phenyl ketone over the patterned area of the master.A poly(dimethylsiloxane) mold is used to confine the liquid PFPE-DMA tothe desired area. The apparatus is then subjected to UV light (λ=365 nm)for 10 minutes while under a nitrogen purge. The fully cured PFPE-DMAmold is then released from the master. The morphology of the mold canthen be confirmed using Atomic Force Microscopy (FIG. 10).

5.6 Fabrication of a Perfluoropolyether-Dimethacrylate (PFPE-DMA) Moldfrom a Template Generated from Earthworm Hemoglobin Protein.

A template, or “master,” for perfluoropolyether-dimethacrylate(PFPE-DMA) mold fabrication is generated by dispersing earthwormhemoglobin proteins on a freshly-cleaved mica surface. This master canbe used to template a patterned mold by pouring PFPE-DMA containing1-hydroxycyclohexyl phenyl ketone over the patterned area of the master.A poly(dimethylsiloxane) mold is used to confine the liquid PFPE-DMA tothe desired area. The apparatus is then subjected to UV light (λ=365 nm)for 10 minutes while under a nitrogen purge. The fully cured PFPE-DMAmold is then released from the master. The morphology of the mold canthen be confirmed using Atomic Force Microscopy.

5.7 Fabrication of a Perfluoropolyether-Dimethacrylate (PFPE-DMA) Moldfrom a Template Generated from Patterned DNA Nanostructures.

A template, or “master,” for perfluoropolyether-dimethacrylate(PFPE-DMA) mold fabrication is generated by dispersing DNAnanostructures on a freshly-cleaved mica surface. This master can beused to template a patterned mold by pouring PFPE-DMA containing1-hydroxycyclohexyl phenyl ketone over the patterned area of the master.A poly(dimethylsiloxane) mold is used to confine the liquid PFPE-DMA tothe desired area. The apparatus is then subjected to UV light (λ=365 nm)for 10 minutes while under a nitrogen purge. The fully cured PFPE-DMAmold is then released from the master. The morphology of the mold canthen be confirmed using Atomic Force Microscopy.

5.8 Fabrication of a Perfluoropolyether-Dimethacrylate (PFPE-DMA) Moldfrom a Template Generated from Carbon Nanotubes

A template, or “master,” for perfluoropolyether-dimethacrylate(PFPE-DMA) mold fabrication is generated by dispersing or growing carbonnanotubes on a silicon oxide wafer. This master can be used to templatea patterned mold by pouring PFPE-DMA containing 1-hydroxycyclohexylphenyl ketone over the patterned area of the master. Apoly(dimethylsiloxane) mold is used to confine the liquid PFPE-DMA tothe desired area. The apparatus is then subjected to UV light (λ=365 nm)for 10 minutes while under a nitrogen purge. The fully cured PFPE-DMAmold is then released from the master. The morphology of the mold canthen be confirmed using Atomic Force Microscopy.

Example 6 Method of Making Monodisperse Nanostructures Having aPlurality of Shapes and Sizes

In some embodiments, the presently disclosed subject matter describes anovel “top down” soft lithographic technique; non-wetting imprintlithography (NoWIL) which allows completely isolated nanostructures tobe generated by taking advantage of the inherent low surface energy andswelling resistance of cured PFPE-based materials.

The presently described subject matter provides a novel “top down” softlithographic technique; non-wetting imprint lithography (NoWIL) whichallows completely isolated nanostructures to be generated by takingadvantage of the inherent low surface energy and swelling resistance ofcured PFPE-based materials. Without being bound to any one particulartheory, a key aspect of NoWIL is that both the elastomeric mold and thesurface underneath the drop of monomer or resin are non-wetting to thisdroplet. If the droplet wets this surface, a thin scum layer willinevitably be present even if high pressures are exerted upon the mold.When both the elastomeric mold and the surface are non-wetting (i.e. aPFPE mold and fluorinated surface) the liquid is confined only to thefeatures of the mold and the scum layer is eliminated as a seal formsbetween the elastomeric mold and the surface under a slight pressure.Thus, the presently disclosed subject matter provides for the first timea simple, general, soft lithographic method to produce nanoparticles ofnearly any material, size, and shape that are limited only by theoriginal master used to generate the mold.

Using NoWIL, nanoparticles composed of 3 different polymers weregenerated from a variety of engineered silicon masters. Representativepatterns include, but are not limited to, 3-μm arrows (see FIG. 17),conical shapes that are 500 nm at the base and converge to <50 nm at thetip (see FIG. 15), and 200-nm trapezoidal structures (see FIG. 13).Definitive proof that all particles were indeed “scum-free” wasdemonstrated by the ability to mechanically harvest these particles bysimply pushing a doctor's blade across the surface. See FIGS. 22 and 24.

Polyethylene glycol (PEG) is a material of interest for drug deliveryapplications because it is readily available, non-toxic, andbiocompatible. The use of PEG nanoparticles generated by inversemicroemulsions to be used as gene delivery vectors has previously beenreported. K. McAllister et al., Journal of the American Chemical Society124, 15198-15207 (Dec. 25, 2002). In the presently disclosed subjectmatter, NoWIL was performed using a commercially availablePEG-diacrylate and blending it with 1 wt % of a photoinitiator,1-hydroxycyclohexyl phenyl ketone. PFPE molds were generated from avariety of patterned silicon substrates using a dimethacrylatefunctionalized PFPE oligomer (PFPE DMA) as described previously. See J.P. Rolland, E. C. Hagberg, G. M. Denison, K. R. Carter, J. M. DeSimone,Angewandte. Chemie-International Edition 43, 5796-5799 (2004). In oneembodiment, flat, uniform, non-wetting surfaces were generated by usinga silicon wafer treated with a fluoroalkyl trichlorosilane or by castinga film of PFPE-DMA on a flat surface and photocuring. A small drop ofPEG diacrylate was then placed on the non-wetting surface and thepatterned PFPE mold placed on top of it. The substrate was then placedin a molding apparatus and a small pressure was applied to push out theexcess PEG-diacrylate. The entire apparatus was then subjected to UVlight (λ=365 nm) for ten minutes while under a nitrogen purge. Particleswere observed after separation of the PFPE mold and flat, non-wettingsubstrate using optical microscopy, scanning electron microscopy (SEM),and atomic force microscopy (AFM).

Poly(lactic acid) (PLA) and derivatives thereof, such aspoly(lactide-co-glycolide) (PLGA), have had a considerable impact on thedrug delivery and medical device communities because it isbiodegradable. See K. E. Uhrich, S. M. Cannizzaro, R. S. Langer, K. M.Shakesheff, Chemical Reviews 99, 3181-3198 (November, 1999); A. C.Albertsson, I. K. Varma, Biomacromolecules 4, 1466-1486(November-December, 2003). As with PEG-based systems, progress has beenmade toward the fabrication of PLGA particles through various dispersiontechniques that result in size distributions and are strictly limited tospherical shapes. See C. Cui, S. P. Schwendeman, Langmuir 34, 8426(2001).

The presently disclosed subject matter demonstrates the use of NoWIL togenerate discrete PLA particles with total control over shape and sizedistribution. For example, in one embodiment, one gram of(3S)-cis-3,6-dimethyl-1,4-dioxane-2,5-dione was heated above its meltingtemperature to 110° C. and ˜20 μL of stannous octoate catalyst/initiatorwas added to the liquid monomer. A drop of the PLA monomer solution wasthen placed into a preheated molding apparatus which contained anon-wetting flat substrate and mold. A small pressure was applied aspreviously described to push out excess PLA monomer. The apparatus wasallowed to heat at 110° C. for 15 h until the polymerization wascomplete. The PFPE-DMA mold and the flat, non-wetting substrate werethen separated to reveal the PLA particles.

To further demonstrate the versatility of NoWIL, particles composed of aconducting polymer polypyrrole (PPy) were generated. PPy particles havebeen formed using dispersion methods, see M. R. Simmons, P. A. Chaloner,S. P. Armes, Langmuir 11, 4222 (1995), as well as “lost-wax” techniques,see P. Jiang, J. F. Bertone, V. L. Colvin, Science 291, 453 (2001).

The presently disclosed subject matter demonstrates for the first time,complete control over shape and size distribution of PPy particles.Pyrrole is known to polymerize instantaneously when in contact withoxidants such as perchloric acid. Dravid et al. has shown that thispolymerization can be retarded by the addition of tetrahydrofuran (THF)to the pyrrole. See M. Su, M. Aslam, L. Fu, N. Q. Wu, V. P. Dravid,Applied Physics Letters 84, 4200-4202 (May 24, 2004).

The presently disclosed subject matter takes advantage of this propertyin the formation of PPy particles by NoWIL. For example, 50 μL of a 1:1v/v solution of THF:pyrrole was added to 50 μL of 70% perchloric acid. Adrop of this clear, brown solution (prior to complete polymerization)into the molding apparatus and applied pressure to remove excesssolution. The apparatus was then placed into the vacuum oven overnightto remove the THF and water. PPy particles were fabricated with goodfidelity using the same masters as previously described.

Importantly, the materials properties and polymerization mechanisms ofPLA, PEG, and PPy are completely different. For example, while PLA is ahigh-modulus, semicrystalline polymer formed using a metal-catalyzedring opening polymerization at high temperature, PEG is a malleable,waxy solid that is photocured free radically, and PPy is a conductingpolymer polymerized using harsh oxidants. The fact that NoWIL can beused to fabricate particles from these diverse classes of polymericmaterials that require very different reaction conditions underscoresits generality and importance.

In addition to its ability to precisely control the size and shape ofparticles, NoWIL offers tremendous opportunities for the facileencapsulation of agents into nanoparticles. As described in Example3-14, NoWIL can be used to encapsulate a 24-mer DNA strand fluorescentlytagged with CY-3 inside the previously described 200 nm trapezoidal PEGparticles. This was accomplished by simply adding the DNA to themonomer/water solution and molding them as described. We were able toconfirm the encapsulation by observing the particles using confocalfluorescence microscopy (see FIG. 30). The presently described approachoffers a distinct advantage over other encapsulation methods in that nosurfactants, condensation agents, and the like are required.Furthermore, the fabrication of monodisperse, 200 nm particlescontaining DNA represents a breakthrough step towards artificialviruses. Accordingly, a host of biologically important agents, such asgene fragments, pharmaceuticals, oligonucleotides, and viruses, can beencapsulated by this method.

The method also is amenable to non-biologically oriented agents, such asmetal nanoparticles, crystals, or catalysts. Further, the simplicity ofthis system allows for straightforward adjustment of particleproperties, such as crosslink density, charge, and composition by theaddition of other comonomers, and combinatorial generation of particleformulations that can be tailored for specific applications.

Accordingly, NoWIL is a highly versatile method for the production ofisolated, discrete nanostructures of nearly any size and shape. Theshapes presented herein were engineered non-arbitrary shapes. NoWIL caneasily be used to mold and replicate non-engineered shapes found innature, such as viruses, crystals, proteins, and the like. Furthermore,the technique can generate particles from a wide variety of organic andinorganic materials containing nearly any cargo. The method issimplistically elegant in that it does not involve complex surfactantsor reaction conditions to generate nanoparticles. Finally, the processcan be amplified to an industrial scale by using existing softlithography roller technology, see Y. N. Xia, D. Qin, G. M. Whitesides,Advanced Materials 8, 1015-1017 (December, 1996), or silk screenprinting methods.

Example 8 Synthesis of Degradable Crosslinkers for Hydrolysable PRINTParticles

Bis(ethylene methacrylate) disulfide (DEDSMA) was synthesized usingmethods described in Li et al. Macromolecules 2005, 38, 8155-8162 from2-hyrdoxyethane disulfide and methacroyl chloride (Scheme 8).Analogously, bis(8-hydroxy-3,6-dioxaoctyl methacrylate) disulfide(TEDSMA) was synthesized from bis(8-hydroxy-3,6-dioxaoctyl) disulfide(Lana et al. Langmuir 1994, 10, 197-210). Methacroyl chloride (0.834 g,8 mmole) was slowly added to a stirred solution ofbis(8-hydroxy-3,6-dioxaoctyl) disulfide (0.662 g, 2 mmole) andtriethylamine (2 mL) in acetonitrile (30 mL) chilled in an ice bath. Thereaction was allowed to warm to room temperature and stirred for 16hours. The mixture was diluted with 5% NaOH solution (50 mL) and stirredfor an additional hour. The mixture was extracted with 2×60 mL ofmethylene chloride, the organic layer was washed 3×100 mL of 1 M NaOH,dried with anhydrous K₂CO₂, and filtered. Removal of the solvent yielded0.860 g of the TEDSMA as a pale yellow oil. ¹H NMR (CDCl₃) δ=6.11 (2H,s), 5.55 (2H, s), 4.29 (4H, t), 3.51-3.8 (16H, m), 2.85 (4H, t), 1.93(6H, s).

8.1 Fabrication of 2 Mm Positively Charged DEDSMA Particles

A patterned perfluoropolyether (PFPE) mold was generated by pouring aPFPE-dimethacrylate (PFPE-DMA) containing 1-hydroxycyclohexyl phenylketone over a silicon substrate patterned with 2 μm rectangles. Apoly(dimethylsiloxane) mold was used to confine the liquid PFPE-DMA tothe desired area. The apparatus was then subjected to UV light λ=365 nm)for 10 minutes while under a nitrogen purge. The fully cured PFPE-DMAmold was then released from the silicon master. Separately, a mixturecomposed of acryloxyethyltrimethylammonium chloride (24.4 mg), DEDSMA(213.0 mg), Polyfluor 570 (2.5 mg), diethoxyacetophenone (5.0 mg),methanol (39.0 mg), acetonitrile (39.0 mg), water (8.0 mg), andN,N-dimethylformamide (6.6 mg) was prepared. This mixture was spotteddirectly onto the patterned PFPE-DMA surface and covered with aseparated unpatterned PFPE-DMA surface. The mold and surface were placedin molding apparatus, purge with N₂ for ten minutes, and placed under atleast 500 N/cm² pressure for 2 hours. The entire apparatus was thensubjected to UV light (λ=365 nm) for 40 minutes while maintainingnitrogen purge. DEDSMA particles were harvested on glass slide usingcyanoacrylate adhesive. The particles were purified by dissolving theadhesive layer with acetone followed by centrifugation of the suspendedparticles (see FIGS. 40 and 41).

8.2 Encapsulation of Calcein Inside 2 μm Positively Charged DEDSMAParticles

A patterned perfluoropolyether (PFPE) mold was generated by pouring aPFPE-dimethacrylate (PFPE-DMA) containing 1-hydroxycyclohexyl phenylketone over a silicon substrate patterned with 2 μm rectangles. Apoly(dimethylsiloxane) mold was used to confine the liquid PFPE-DMA tothe desired area. The apparatus was then subjected to UV light (λ=365nm) for 10 minutes while under a nitrogen purge. The fully curedPFPE-DMA mold was then released from the silicon master. Separately, amixture composed of acryloxyethyltrimethylammonium chloride (3.4 mg),DEDSMA (29.7 mg), calcein (0.7 mg), Polyfluor 570 (0.35 mg),diethoxyacetophenone (0.7 mg), methanol (5.45 mg), acetonitrile (5.45mg), water (1.11 mg), and N,N-dimethylformamide (6.6 mg) was prepared.This mixture was spotted directly onto the patterned PFPE-DMA surfaceand covered with a separated unpatterned PFPE-DMA surface. The mold andsurface were placed in molding apparatus, purge with N₂ for ten minutes,and placed under at least 500 N/cm² pressure for 2 hours. The entireapparatus was then subjected to UV light (λ=365 nm) for 40 minutes whilemaintaining nitrogen purge. Calcein containing DEDSMA particles wereharvested on glass slide using cyanoacrylate adhesive. The particleswere purified by dissolving the adhesive layer with acetone followed bycentrifugation of the suspended particles (see FIG. 42).

8.3 Encapsulation of Plasmid DNA into Charged DEDSMA Particles

A patterned perfluoropolyether (PFPE) mold was generated by pouring aPFPE-dimethacrylate (PFPE-DMA) containing 1-hydroxycyclohexyl phenylketone over a silicon substrate patterned with 2 μm rectangles. Apoly(dimethylsiloxane) mold was used to confine the liquid PFPE-DMA tothe desired area. The apparatus was then subjected to UV light (λ=365nm) for 10 minutes while under a nitrogen purge. The fully curedPFPE-DMA mold was then released from the silicon master. Separately, 0.5μg of fluorescein-labelled plasmid DNA (Mirus Biotech) as a 0.25 μg/μLsolution in TE buffer and a 2.0 μg of pSV β-galactosidase control vector(Promega) as a 1.0 μg/μL solution in TE buffer were sequentially addedto a mixture composed of acryloxyethyltrimethylammonium chloride (1.44mg), DEDSMA (12.7 mg), Polyfluor 570 (Polysciences, 0.08 mg),1-hydroxycyclohexyl phenyl ketone (0.28 mg), methanol (5.96 mg),acetonitrile (5.96 mg), water (0.64 mg), and N,N-dimethylformamide(14.16 mg). This mixture was spotted directly onto the patternedPFPE-DMA surface and covered with a separated unpatterned PFPE-DMAsurface. The mold and surface were placed in molding apparatus, purgewith N₂ for ten minutes, and placed under at least 500 N/cm² pressurefor 2 hours. The entire apparatus was then subjected to UV light (λ=365nm) for 40 minutes while maintaining nitrogen purge. These particleswere harvested on glass slide using cyanoacrylate adhesive. Theparticles were purified by dissolving the adhesive layer with acetonefollowed by centrifugation of the suspended particles (see FIG. 43).

8.4 Encapsulation of Plasmid DNA into PEG Particles

A patterned perfluoropolyether (PFPE) mold was generated by pouring aPFPE-dimethacrylate (PFPE-DMA) containing 1-hydroxycyclohexyl phenylketone over a silicon substrate patterned with 2 μm rectangles. Apoly(dimethylsiloxane) mold was used to confine the liquid PFPE-DMA tothe desired area. The apparatus was then subjected to UV light (λ=365nm) for 10 minutes while under a nitrogen purge. The fully curedPFPE-DMA mold was then released from the silicon master. Separately, 0.5μg of fluorescein-labelled plasmid DNA (Mirus Biotech) as a 0.25 μg/Lsolution in TE buffer and a 2.0 μg of pSV β-galactosidase control vector(Promega) as a 1.0 μg/μL solution in TE buffer were sequentially addedto a mixture composed of acryloxyethyltrimethylammonium chloride (1.2mg), polyethylene glycol diacrylate (n=9) (10.56 mg), Polyfluor 570(Polysciences, 0.12 mg), diethoxyacetophenone (0.12 mg), methanol (1.5mg), water (0.31 mg), and N,N-dimethylformamide (7.2 mg). This mixturewas spotted directly onto the patterned PFPE-DMA surface and coveredwith a separated unpatterned PFPE-DMA surface. The mold and surface wereplaced in molding apparatus, purge with N₂ for ten minutes, and placedunder at least 500 N/cm² pressure for 2 hours. The entire apparatus wasthen subjected to UV light (λ=365 nm) for 40 minutes while maintainingnitrogen purge. These particles were harvested on glass slide usingcyanoacrylate adhesive. The particles were purified by dissolving theadhesive layer with acetone followed by centrifugation of the suspendedparticles (see FIG. 44).

The following references may provide information and techniques tosupplement some of the techniques and parameters of the presentexamples, therefore, the references are incorporated by reference hereinin their entirety including any and all references cited therein. Li,Y., and Armes, S. P. Synthesis and Chemical Degradation of BranchedVinyl Polymers Prepared via ATRP: Use of a Cleavable Disulfide-BasedBranching Agent. Macromolecules 2005; 38: 8155-8162; and Lang, H.,Duschl, C., and Vogel, H. (1994), A new class of thiolipids for theattachment of lipid bilayers on gold surfaces. Langmuir 10, 197-210.

Example 9 Cellular Uptake of PRINT Particles Effect of Charge

9.1 Fabrication of 200 nm Cylindrical Fluorescently-Tagged Neutral PEGParticles

A patterned perfluoropolyether (PFPE) mold is generated by pouring aPFPE-dimethacrylate (PFPE-DMA) containing 2,2-diethoxyacetophenone overa silicon substrate patterned with 200 nm cylindrical shapes (see FIG.45). The apparatus is then subjected to a nitrogen purge for 10 minutesbefore the application of UV light (λ=365 nm) for 10 minutes while undera nitrogen purge. The fully cured PFPE-DMA mold is then released fromthe silicon master. Separately, a poly(ethylene glycol) (PEG) diacrylate(n=9) is blended with 28 wt % PEG methacrylate (n=9), 2 wt %azobisisobutyronitrile (AIBN), and 0.25 wt % rhodamine methacrylate.Flat, uniform, non-wetting surfaces are generated by coating a glassslide with PFPE-dimethacrylate (PFPE-DMA) containing2,2-diethoxyacetophenone. The slide is then subjected to a nitrogenpurge for 10 minutes, then UV light is applied (λ=365 nm) while under anitrogen purge. The flat, fully cured PFPE-DMA substrate is releasedfrom the slide. Following this, 0.1 mL of the monomer blend is evenlyspotted onto the flat PFPE-DMA surface and then the patterned PFPE-DMAmold placed on top of it. The surface and mold are then placed in amolding apparatus and a small amount of pressure is applied to removeany excess monomer solution. The entire apparatus is purged withnitrogen for 10 minutes, then subjected to UV light (λ=365 nm) for 10minutes while under a nitrogen purge. Neutral PEG nanoparticles areobserved after separation of the PFPE-DMA mold and substrate usingscanning electron microscopy (SEM). The harvesting process begins byspraying a thin layer of cyanoacrylate monomer onto the PFPE-DMA moldfilled with particles. The PFPE-DMA mold is immediately placed onto aglass slide and the cyanoacrylate is allowed to polymerize in an anionicfashion for one minute. The mold is removed and the particles areembedded in the soluble adhesive layer (see FIG. 46), which providesisolated, harvested colloidal particle dispersions upon dissolution ofthe soluble adhesive polymer layer in acetone. Particles embedded in theharvesting layer, or dispersed in acetone can be visualized by SEM. Thedissolved poly(cyanoacrylate) can remain with the particles in solution,or can be removed via centrifugation.

9.2 Fabrication of 200 nm Cylindrical Fluorescently-Tagged 14 wt %Cationically Charged PEG Particles

A patterned perfluoropolyether (PFPE) mold is generated by pouring aPFPE-dimethacrylate (PFPE-DMA) containing 2,2-diethoxyacetophenone overa silicon substrate patterned with 200 nm cylindrical shapes (see FIG.45). The apparatus is then subjected to a nitrogen purge for 10 minutesbefore the application of UV light (λ=365 nm) for 10 minutes while undera nitrogen purge. The fully cured PFPE-DMA mold is then released fromthe silicon master. Separately, a poly(ethylene glycol) (PEG) diacrylate(n=9) is blended with 14 wt % PEG methacrylate (n=9), 14 wt %2-acryloxyethyltrimethylammonium chloride (AETMAC), 2 wt %azobisisobutyronitrile (AIBN), and 0.25 wt % rhodamine methacrylate.Flat, uniform, non-wetting surfaces are generated by coating a glassslide with PFPE-dimethacrylate (PFPE-DMA) containing2,2-diethoxyacetophenone. The slide is then subjected to a nitrogenpurge for 10 minutes, then UV light is applied (λ=365 nm) while under anitrogen purge. The flat, fully cured PFPE-DMA substrate is releasedfrom the slide. Following this, 0.1 mL of the monomer blend is evenlyspotted onto the flat PFPE-DMA surface and then the patterned PFPE-DMAmold placed on top of it. The surface and mold are then placed in amolding apparatus and a small amount of pressure is applied to removeany excess monomer solution. The entire apparatus is purged withnitrogen for 10 minutes, then subjected to UV light (λ=365 nm) for 10minutes while under a nitrogen purge. Cationically charged PEGnanoparticles are observed after separation of the PFPE-DMA mold andsubstrate using scanning electron microscopy (SEM). The harvestingprocess begins by spraying a thin layer of cyanoacrylate monomer ontothe PFPE-DMA mold filled with particles. The PFPE-DMA mold isimmediately placed onto a glass slide and the cyanoacrylate is allowedto polymerize in an anionic fashion for one minute. The mold is removedand the particles are embedded in the soluble adhesive layer (see FIG.46), which provides isolated, harvested colloidal particle dispersionsupon dissolution of the soluble adhesive polymer layer in acetone.Particles embedded in the harvesting layer or dispersed in acetone canbe visualized by SEM. The dissolved poly(cyanoacrylate) can remain withthe particles in solution, or can be removed via centrifugation.

9.3 Fabrication of 200 nm Cylindrical Fluorescently-Tagged 28 wt %Cationically Charged PEG Particles

A patterned perfluoropolyether (PFPE) mold is generated by pouring aPFPE-dimethacrylate (PFPE-DMA) containing 2,2-diethoxyacetophenone overa silicon substrate patterned with 200 nm cylindrical shapes (see FIG.45). The apparatus is then subjected to a nitrogen purge for 10 minutesbefore the application of UV light (λ=365 nm) for 10 minutes while undera nitrogen purge. The fully cured PFPE-DMA mold is then released fromthe silicon master. Separately, a poly(ethylene glycol) (PEG) diacrylate(n=9) is blended with 28 wt % 2-acryloxyethyltrimethylammonium chloride(AETMAC), 2 wt % azobisisobutyronitrile (AIBN), and 0.25 wt % rhodaminemethacrylate. Flat, uniform, non-wetting surfaces are generated bycoating a glass slide with PFPE-dimethacrylate (PFPE-DMA) containing2,2-diethoxyacetophenone. The slide is then subjected to a nitrogenpurge for 10 minutes, then UV light is applied (λ=365 nm) while under anitrogen purge. The flat, fully cured PFPE-DMA substrate is releasedfrom the slide. Following this, 0.1 mL of the monomer blend is evenlyspotted onto the flat PFPE-DMA surface and then the patterned PFPE-DMAmold placed on top of it. The surface and mold are then placed in amolding apparatus and a small amount of pressure is applied to removeany excess monomer solution. The entire apparatus is purged withnitrogen for 10 minutes, then subjected to UV light (λ=365 nm) for 10minutes while under a nitrogen purge. Cationically charged PEGnanoparticles are observed after separation of the PFPE-DMA mold andsubstrate using scanning electron microscopy (SEM). The harvestingprocess begins by spraying a thin layer of cyanoacrylate monomer ontothe PFPE-DMA mold filled with particles. The PFPE-DMA mold isimmediately placed onto a glass slide and the cyanoacrylate is allowedto polymerize in an anionic fashion for one minute. The mold is removedand the particles are embedded in the soluble adhesive layer (see FIG.46), which provides isolated, harvested colloidal particle dispersionsupon dissolution of the soluble adhesive polymer layer in acetone.Particles embedded in the harvesting layer or dispersed in acetone canbe visualized by SEM. The dissolved poly(cyanoacrylate) can remain withthe particles in solution, or can be removed via centrifugation.

9.4 Cellular Uptake of 200 nm Cylindrically Shaped Neutral PEG PRINTParticles

The neutral 200 nm cylindrical PEG particles (aspect ratio=1:1, 200nm×200 nm particles) fabricated using PRINT were dispersed in 250 μL ofwater to be used in cellular uptake experiments. These particles wereexposed to NIH 3T3 (mouse embryonic) cells at a final concentration ofparticles of 60 μg/mL. The particles and cells were incubated for 4 hrsat 5% CO₂ at 37° C. The cells were then characterized via confocalmicroscopy (see FIG. 47) and cell toxicities were assessed using an MTTassay (see FIG. 48),

9.5 Cellular Uptake of 200 nm Cylindrically Shaped 14 Wt % CationicallyCharged PEG PRINT Particles

The 14 wt % cationically charged 200 nm cylindrical PEG particles(aspect ratio=1:1, 200 nm×200 nm particles) fabricated using PRINT weredispersed in 250 μL of water to be used in cellular uptake experiments.These particles were exposed to NIH 3T3 (mouse embryonic) cells at afinal concentration of particles of 60 μg/mL. The particles and cellswere incubated for 4 hrs at 5% CO₂ at 37° C. The cells were thencharacterized via confocal microscopy (see FIG. 47) and cell toxicitieswere assessed using an MTT assay (see FIG. 48).

9.6 Cellular Uptake of 200 nm Cylindrically Shaped 28 Wt % CationicallyCharged PEG PRINT Particles

The 28 wt % cationically charged 200 nm cylindrical PEG particles(aspect ratio=1:1, 200 nm×200 nm particles) fabricated using PRINT weredispersed in 250 μL of water to be used in cellular uptake experiments.These particles were exposed to NIH 3T3 (mouse embryonic) cells at afinal concentration of particles of 60 μg/mL. The particles and cellswere incubated for 4 hrs at 5% CO₂ at 37° C. The cells were thencharacterized via confocal microscopy (see FIG. 47) and cell toxicitieswere assessed using an MTT assay (see FIG. 48).

Example 10 Cellular Uptake of PRINT Particles Effect of Size

10.1 Fabrication of 200 nm Cylindrical Fluorescently-Tagged 14 wt %Cationically Charged PEG Particles—Repeat

A patterned perfluoropolyether (PFPE) mold is generated by pouring aPFPE-dimethacrylate (PFPE-DMA) containing 2,2-diethoxyacetophenone overa silicon substrate patterned with 200 nm cylindrical shapes (see FIG.45). The apparatus is then subjected to a nitrogen purge for 10 minutesbefore the application of UV light (λ=365 nm) for 10 minutes while undera nitrogen purge. The fully cured PFPE-DMA mold is then released fromthe silicon master. Separately, a poly(ethylene glycol) (PEG) diacrylate(n=9) is blended with 14 wt % PEG methacrylate (n=9), 14 wt %2-acryloxyethyltrimethylammonium chloride (AETMAC), 2 wt %azobisisobutyronitrile (AIBN), and 0.25 wt % rhodamine methacrylate.Flat, uniform, non-wetting surfaces are generated by coating a glassslide with PFPE-dimethacrylate (PFPE-DMA) containing2,2-diethoxyacetophenone. The slide is then subjected to a nitrogenpurge for 10 minutes, then UV light is applied (λ=365 nm) while under anitrogen purge. The flat, fully cured PFPE-DMA substrate is releasedfrom the slide. Following this, 0.1 mL of the monomer blend is evenlyspotted onto the flat PFPE-DMA surface and then the patterned PFPE-DMAmold placed on top of it. The surface and mold are then placed in amolding apparatus and a small amount of pressure is applied to removeany excess monomer solution. The entire apparatus is purged withnitrogen for 10 minutes, then subjected to UV light (λ=365 nm) for 10minutes while under a nitrogen purge. Cationically charged PEGnanoparticles are observed after separation of the PFPE-DMA mold andsubstrate using scanning electron microscopy (SEM). The harvestingprocess begins by spraying a thin layer of cyanoacrylate monomer ontothe PFPE-DMA mold filled with particles. The PFPE-DMA mold isimmediately placed onto a glass slide and the cyanoacrylate is allowedto polymerize in an anionic fashion for one minute. The mold is removedand the particles are embedded in the soluble adhesive layer (see FIG.46), which provides isolated, harvested colloidal particle dispersionsupon dissolution of the soluble adhesive polymer layer in acetone.Particles embedded in the harvesting layer or dispersed in acetone canbe visualized by SEM. The dissolved poly(cyanoacrylate) can remain withthe particles in solution, or can be removed via centrifugation.

10.2 Fabrication of 2 μm×2 μm×1 μm Cubic Fluorescently-tagged 14 wt %Cationically Charged PEG Particles

A patterned perfluoropolyether (PFPE) mold is generated by pouring aPFPE-dimethacrylate (PFPE-DMA) containing 2,2-diethoxyacetophenone overa silicon substrate patterned with 2 μm×2 μm×1 μm cubic shapes. Theapparatus is then subjected to a nitrogen purge for 10 minutes beforethe application of UV light (λ=365 nm) for 10 minutes while under anitrogen purge. The fully cured PFPE-DMA mold is then released from thesilicon master. Separately, a poly(ethylene glycol) (PEG) diacrylate(n=9) is blended with 14 wt % PEG methacrylate (n=9), 14 wt %2-acryloxyethyltrimethylammonium chloride (AETMAC), 2 wt %azobisisobutyronitrile (AIBN), and 0.25 wt % rhodamine methacrylate.Flat, uniform, non-wetting surfaces are generated by coating a glassslide with PFPE-dimethacrylate (PFPE-DMA) containing2,2-diethoxyacetophenone. The slide is then subjected to a nitrogenpurge for 10 minutes, then UV light is applied (λ=365 nm) while under anitrogen purge. The flat, fully cured PFPE-DMA substrate is releasedfrom the slide. Following this, 0.1 mL of the monomer blend is evenlyspotted onto the flat PFPE-DMA surface and then the patterned PFPE-DMAmold placed on top of it. The surface and mold are then placed in amolding apparatus and a small amount of pressure is applied to removeany excess monomer solution. The entire apparatus is purged withnitrogen for 10 minutes, then subjected to UV light (λ=365 nm) for 10minutes while under a nitrogen purge. Cationically charged PEGnanoparticles are observed after separation of the PFPE-DMA mold andsubstrate using scanning electron microscopy (SEM), optical andfluorescence microscopy (excitation λ=526 nm, emission λ=555 nm). Theharvesting process begins by spraying a thin layer of cyanoacrylatemonomer onto the PFPE-DMA mold filled with particles. The PFPE-DMA moldis immediately placed onto a glass slide and the cyanoacrylate isallowed to polymerize in an anionic fashion for one minute. The mold isremoved and the particles are embedded in the soluble adhesive layer,which provides isolated, harvested colloidal particle dispersions upondissolution of the soluble adhesive polymer layer in acetone. Particlesembedded in the harvesting layer or dispersed in acetone can bevisualized by SEM. The dissolved poly(cyanoacrylate) can remain with theparticles in solution, or can be removed via centrifugation.

10.3 Fabrication of 5 μm×5 μm×5 μm Cubic Fluorescently-Tagged 14 wt %Cationically Charged PEG Particles

A patterned perfluoropolyether (PFPE) mold is generated by pouring aPFPE-dimethacrylate (PFPE-DMA) containing 2,2-diethoxyacetophenone overa silicon substrate patterned with 5 μm×5 μm×5 μm cubic shapes. Theapparatus is then subjected to a nitrogen purge for 10 minutes beforethe application of UV light (λ=365 nm) for 10 minutes while under anitrogen purge. The fully cured PFPE-DMA mold is then released from thesilicon master. Separately, a poly(ethylene glycol) (PEG) diacrylate(n=9) is blended with 14 wt % PEG methacrylate (n=9), 14 wt %2-acryloxyethyltrimethylammonium chloride (AETMAC), 2 wt %azobisisobutyronitrile (AIBN), and 0.25 wt % rhodamine methacrylate.Flat, uniform, non-wetting surfaces are generated by coating a glassslide with PFPE-dimethacrylate (PFPE-DMA) containing2,2-diethoxyacetophenone. The slide is then subjected to a nitrogenpurge for 10 minutes, then UV light is applied (λ=365 nm) while under anitrogen purge. The flat, fully cured PFPE-DMA substrate is releasedfrom the slide. Following this, 0.1 mL of the monomer blend is evenlyspotted onto the flat PFPE-DMA surface and then the patterned PFPE-DMAmold placed on top of it. The surface and mold are then placed in amolding apparatus and a small amount of pressure is applied to removeany excess monomer solution. The entire apparatus is purged withnitrogen for 10 minutes, then subjected to UV light (λ=365 nm) for 10minutes while under a nitrogen purge. Cationically charged PEGnanoparticles are observed after separation of the PFPE-DMA mold andsubstrate using scanning electron microscopy (SEM), optical andfluorescence microscopy (excitation λ=526 nm, emission λ=555 nm). Theharvesting process begins by spraying a thin layer of cyanoacrylatemonomer onto the PFPE-DMA mold filled with particles. The PFPE-DMA moldis immediately placed onto a glass slide and the cyanoacrylate isallowed to polymerize in an anionic fashion for one minute. The mold isremoved and the particles are embedded in the soluble adhesive layer,which provides isolated, harvested colloidal particle dispersions upondissolution of the soluble adhesive polymer layer in acetone. Particlesembedded in the harvesting layer, or dispersed in acetone can bevisualized by SEM. The dissolved poly(cyanoacrylate) can remain with theparticles in solution, or can be removed via centrifugation.

10.4 Cellular Uptake of 200 nm Cylindrically Shaped 14 Wt % CationicallyCharged PEG PRINT Particles—Repeat

The 14 wt % cationically charged 200 nm cylindrical PEG particles(aspect ratio=1:1, 200 nm×200 nm particles) fabricated using PRINT weredispersed in 250 μL of water to be used in cellular uptake experiments.These particles were exposed to NIH 3T3 (mouse embryonic) cells at afinal concentration of particles of 60 μg/mL. The particles and cellswere incubated for 4 hrs at 5% CO₂ at 37° C. The cells were thencharacterized via confocal microscopy (see FIG. 49).

10.5 Cellular Uptake of 2 μm×2 μm×1 μm Cubic Shaped 14 wt % CationicallyCharged PEG PRINT Particles

The 14 wt % cationically charged 2 μm×2 μm×1 μm cubic PEG particlesfabricated using PRINT were dispersed in 250 μL of water to be used incellular uptake experiments. These particles were exposed to NIH 3T3(mouse embryonic) cells at a final concentration of particles of 60μg/mL. The particles and cells were incubated for 4 hrs at 5% CO₂ at 37°C. The cells were then characterized via confocal microscopy (see FIG.49).

10.6 Cellular Uptake of 5 μm×5 μm×5 μm Cubic Shaped 14 wt % CationicallyCharged PEG PRINT Particles

The 14 wt % cationically charged 5 μm×5 μm×5 μm cubic PEG particlesfabricated using PRINT were dispersed in 250 μL of water to be used incellular uptake experiments. These particles were exposed to NIH 3T3(mouse embryonic) cells at a final concentration of particles of 60μg/mL. The particles and cells were incubated for 4 hrs at 5% CO₂ at 37°C. The cells were then characterized via confocal microscopy (see FIG.49).

Example 11 Cellular Uptake of DEDSMA PRINT Particles

11.1 Cellular Uptake of DEDSMA PRINT Particles

The DEDSMA particles fabricated using PRINT were dispersed in 250 μL ofwater to be used in cellular uptake experiments. These particles wereexposed to NIH 3T3 (mouse embryonic) cells at a final concentration ofparticles of 60 μg/mL. The particles and cells were incubated for 4 hrsat 5% CO₂ at 37° C. The cells were then characterized via confocalmicroscopy.

Example 12 Radiolabeling PRINT Particles

12.1 Synthesis of ¹⁴C radiolabeled 2 μm×2 μm×1 μm Cubic PRINT Particles

A patterned perfluoropolyether (PFPE) mold is generated by pouring aPFPE-dimethacrylate (PFPE-DMA) containing 2,2-diethoxyacetophenone overa silicon substrate patterned with 2 μm×2 μm×1 μm cubic shapes. Theapparatus is then subjected to a nitrogen purge for 10 minutes beforethe application of UV light (λ=365 nm) for 10 minutes while under anitrogen purge. The fully cured PFPE-DMA mold is then released from thesilicon master. Separately, a poly(ethylene glycol) (PEG) diacrylate(n=9) is blended with 30 wt % 2-aminoethylmethacrylate hydrochloride(AEM), and 1 wt % 2,2-diethoxyacetophenone. The monomer solution isapplied to the mold by spraying a diluted (10×) blend of the monomerswith isopropyl alcohol. A polyethylene sheet is placed onto the mold,and any residual air bubbles are pushed out with a roller. The sheet isslowly pulled back from the mold at a rate of 1 inch/minute. The mold isthen subjected to a nitrogen purge for 10 minutes, then UV light isapplied (λ=365 nm) while under a nitrogen purge. The harvesting processbegins by spraying a thin layer of cyanoacrylate monomer onto thePFPE-DMA mold filled with particles. The PFPE-DMA mold is immediatelyplaced onto a glass slide and the cyanoacrylate is allowed to polymerizein an anionic fashion for one minute. The mold is removed and theparticles are embedded in the soluble adhesive layer, which providesisolated, harvested colloidal particle dispersions upon dissolution ofthe soluble adhesive polymer layer in acetone. Particles embedded in theharvesting layer, or dispersed in acetone can be visualized by SEM, andoptical microscopy. The dissolved poly(cyanoacrylate) can remain withthe particles in solution, or can be removed via centrifugation. Thedry, purified particles are then exposed to ¹⁴C-acetic anhydride in drydichloromethane in the presence of triethylamine, and4-dimethylaminopyridine for 24 hours (see FIG. 50). Unreacted reagentsare removed via centrifugation. Efficiency of the reaction is monitoredby measured the emitted radioactivity in a scintillation vial.

12.2 Synthesis of ¹⁴C Radiolabeled 200 nm Cylindrical PRINT Particles

A patterned perfluoropolyether (PFPE) mold is generated by pouring aPFPE-dimethacrylate (PFPE-DMA) containing 2,2-diethoxyacetophenone overa silicon substrate patterned with 200 nm cylindrical shapes. Theapparatus is then subjected to a nitrogen purge for 10 minutes beforethe application of UV light (λ=365 nm) for 10 minutes while under anitrogen purge. The fully cured PFPE-DMA mold is then released from thesilicon master. Separately, a poly(ethylene glycol) (PEG) diacrylate(n=9) is blended with 30 wt % 2-aminoethylmethacrylate hydrochloride(AEM), and 1 wt % 2,2-diethoxyacetophenone. The monomer solution isapplied to the mold by spraying a diluted (10×) blend of the monomerswith isopropyl alcohol. A polyethylene sheet is placed onto the mold,and any residual air bubbles are pushed out with a roller. The sheet isslowly pulled back from the mold at a rate of 1 inch/minute. The mold isthen subjected to a nitrogen purge for 10 minutes, then UV light isapplied (λ=365 nm) while under a nitrogen purge. The harvesting processbegins by spraying a thin layer of cyanoacrylate monomer onto thePFPE-DMA mold filled with particles. The PFPE-DMA mold is immediatelyplaced onto a glass slide and the cyanoacrylate is allowed to polymerizein an anionic fashion for one minute. The mold is removed and theparticles are embedded in the soluble adhesive layer, which providesisolated, harvested colloidal particle dispersions upon dissolution ofthe soluble adhesive polymer layer in acetone. Particles embedded in theharvesting layer, or dispersed in acetone can be visualized by SEM. Thedissolved poly(cyanoacrylate) can remain with the particles in solution,or can be removed via centrifugation. The dry, purified particles arethen exposed to ¹⁴C-acetic anhydride in dry dichloromethane in thepresence of triethylamine, and 4-dimethylaminopyridine for 24 hours (seeFIG. 50). Unreacted reagents are removed via centrifugation. Efficiencyof the reaction is monitored by measured the emitted radioactivity in ascintillation vial.

12.3 Fabrication of Pendant Gadolinium PEG Particles

A patterned perfluoropolyether (PFPE) mold is generated by pouring aPFPE-dimethacrylate (PFPE-DMA) containing 2,2′-diethoxy-acetophenoneover a silicon substrate patterned with 3×3×11 um pillar shapes. Theapparatus is then subjected to UV light (λ=365 nm) for 15 minutes whileunder a nitrogen purge. The fully cured PFPE-DMA mold is then releasedfrom the silicon master. Separately, a poly(ethylene glycol) (PEG)diacrylate (n=9) is blended with 1 wt % of a photoinitiator,2,2′-diethoxy-acetophenone. 20 μL of chloroform, 70 μL of PEG diacrylatemonomer and 30 uL of DPTA-PEG-acrylate are mixed. Flat, uniform,non-wetting surfaces are generated by pouring a PFPE-dimethacrylate(PFPE-DMA) containing 2,2′-diethoxy-acetophenone over a silicon waferand then subjected to UV light (λ=365 nm) for 15 minutes while under anitrogen purge. Following this, 50 μL of the PEG diacrylate solution isthen placed on the non-wetting surface and the patterned PFPE moldplaced on top of it. The substrate is then placed in a molding apparatusand a small pressure is applied to push out excess PEG-diacrylatesolution. The entire apparatus is then subjected to UV light (λ=365 nm)for 15 minutes while under a nitrogen purge. Particles are observedafter separation of the PFPE mold. The particles were harvestedutilizing a sacrificial adhesive layer and verified via DIC microscopy.These particles were subsequently treated with an aqueous solution ofGd(NO₃)₃. These particles were then dispersed in a agrose gel and TIweighted imaging profiles were examined utilizing a Siemens Allegra 3Thead magnetic resonance instrument (see FIG. 51).

12.4 Forming a Particle Containing CDI Linker

A patterned perfluoropolyether (PFPE) mold is generated by pouring aPFPE-dimethacrylate (PFPE-DMA) containing 2,2′-diethoxy-acetophenoneover a silicon substrate patterned with 200 nm shapes. The apparatus isthen subjected to UV light (λ=365 nm) for 15 minutes while under anitrogen purge. The fully cured PFPE-DMA mold is then released from thesilicon master. Separately, a poly(ethylene glycol) (PEG) diacrylate(n=9) is blended with 1 wt % of a photoinitiator,2,2′-diethoxy-acetophenone. 70 μL of PEG diacrylate monomer and 30 uL ofCDI-PEG monomer were mixed. Specifically, the CDI-PEG monomer wassynthesized by adding 1,1′-carbonyl diimidazole (CDI) to a solution ofPEG (n=400) monomethylacrylate in chloroform. This solution was allowedto stir overnight. This solution was then further purified by anextraction with cold water. The resulting CDI-PEG monomethacrylate wasthen isolated via vacuum. Flat, uniform, non-wetting surfaces aregenerated by pouring a PFPE-dimethacrylate (PFPE-DMA) containing2,2′-diethoxy-acetophenone over a silicon wafer and then subjected to UVlight (μ=365 nm) for 15 minutes while under a nitrogen purge. Followingthis, 50 μL of the PEG diacrylate solution is then placed on the nonwetting surface and the patterned PFPE mold placed on top of it. Thesubstrate is then placed in a molding apparatus and a small pressure isapplied to push out excess PEG-diacrylate solution. The entire apparatusis then subjected to UV light (h=365 nm) for 15 minutes while under anitrogen purge. Particles are observed after separation of the PFPEmold. The particles were harvested utilizing a sacrificial adhesivelayer and verified via DIC microscopy. This linker can be utilized toattach an amine containing target onto the particle (see FIG. 52).

12.5 Tethering Avidin to the CDI Linker

A patterned perfluoropolyether (PFPE) mold is generated by pouring aPFPE-dimethacrylate (PFPE-DMA) containing 2,2′-diethoxy-acetophenoneover a silicon substrate patterned with 200 nm shapes. The apparatus isthen subjected to UV light (λ=365 nm) for 15 minutes while under anitrogen purge. The fully cured PFPE-DMA mold is then released from thesilicon master. Separately, a poly(ethylene glycol) (PEG) diacrylate(n=9) is blended with 1 wt % of a photoinitiator,2,2′-diethoxy-acetophenone. 70 μL of PEG diacrylate monomer and 30 uL ofCDI-PEG monomer were mixed. Specifically, the CDI-PEG monomer wassynthesized by adding 1,1′-carbonyl diimidazole (CDI) to a solution ofPEG (n=400) monomethylacrylate in chloroform. This solution was allowedto stir overnight. This solution was then further purified by anextraction with cold water. The resulting CDI-PEG monomethacrylate wasthen isolated via vacuum. Flat, uniform, non-wetting surfaces aregenerated by pouring a PFPE-dimethacrylate (PFPE-DMA) containing2,2′-diethoxy-acetophenone over a silicon wafer and then subjected to UVlight (λ=365 nm) for 15 minutes while under a nitrogen purge. Followingthis, 50 μL of the PEG diacrylate solution is then placed on the nonwetting surface and the patterned PFPE mold placed on top of it. Thesubstrate is then placed in a molding apparatus and a small pressure isapplied to push out excess PEG-diacrylate solution. The entire apparatusis then subjected to UV light (λ=365 nm) for 15 minutes while under anitrogen purge. Particles are observed after separation of the PFPEmold. The particles were harvested utilizing a sacrificial adhesivelayer and verified via DIC microscopy. These particles containing theCDI linker group were subsequently treated with and aqueous solution offluorescently tagged avidin. These particles were allowed to stir atroom temperature for four hours. These particles were then isolated viacentrifugation and rinsed with deionized water. Attachment was confirmedvia confocal microscopy (see FIG. 53).

12.6 Fabrication of PEG Particles that Target the HER2 Receptor

A patterned perfluoropolyether (PFPE) mold is generated by pouring aPFPE-dimethacrylate (PFPE-DMA) containing 2,2′-diethoxy-acetophenoneover a silicon substrate patterned with 200 nm shapes. The apparatus isthen subjected to UV light (λ=365 nm) for 15 minutes while under anitrogen purge. The fully cured PFPE-DMA mold is then released from thesilicon master. Separately, a poly(ethylene glycol) (PEG) diacrylate(n=9) is blended with 1 wt % of a photoinitiator,2,2′-diethoxy-acetophenone. 70 μL of PEG diacrylate monomer and 30 uL ofCDI-PEG monomer were mixed. Specifically, the CDI-PEG monomer wassynthesized by adding 1,1′-carbonyl diimidazole (CDI) to a solution ofPEG (n=400) monomethylacrylate in chloroform. This solution was allowedto stir overnight. This solution was then further purified by anextraction with cold water. The resulting CDI-PEG monomethacrylate wasthen isolated via vacuum. Flat, uniform, non-wetting surfaces aregenerated by pouring a PFPE-dimethacrylate (PFPE-DMA) containing2,2′-diethoxy-acetophenone over a silicon wafer and then subjected to UVlight (λ=365 nm) for 15 minutes while under a nitrogen purge. Followingthis, 50 μL of the PEG diacrylate solution is then placed on the nonwetting surface and the patterned PFPE mold placed on top of it. Thesubstrate is then placed in a molding apparatus and a small pressure isapplied to push out excess PEG-diacrylate solution. The entire apparatusis then subjected to UV light (λ=365 nm) for 15 minutes while under anitrogen purge. Particles are observed after separation of the PFPEmold. The particles were harvested utilizing a sacrificial adhesivelayer and verified via DIC microscopy. These particles containing theCDI linker group were subsequently treated with and aqueous solution offluorescently tagged avidin. These particles were allowed to stir atroom temperature for four hours. These particles were then isolated viacentrifugation and rinsed with deionized water. These avidin labeledparticles were then treated with biotinylated FAB fragments. Attachmentwas confirmed via confocal microscopy (see FIG. 54).

12.7 Fabrication of PEG Particles that Target Non-Hodgkin's Lymphoma

A patterned perfluoropolyether (PFPE) mold is generated by pouring aPFPE-dimethacrylate (PFPE-DMA) containing 2,2′-diethoxy-acetophenoneover a silicon substrate patterned with 200 nm shapes. The apparatus isthen subjected to UV light (λ=365 nm) for 15 minutes while under anitrogen purge. The fully cured PFPE-DMA mold is then released from thesilicon master. Separately, a poly(ethylene glycol) (PEG) diacrylate(n=9) is blended with 1 wt % of a photoinitiator,2,2′-diethoxy-acetophenone. 70 μL of PEG diacrylate monomer and 30 uL ofCDI-PEG monomer were mixed. Specifically, the CDI-PEG monomer wassynthesized by adding 1,1′-carbonyl diimidazole (CDI) to a solution ofPEG (n=400) monomethylacrylate in chloroform. This solution was allowedto stir overnight. This solution was then further purified by anextraction with cold water. The resulting CDI-PEG monomethacrylate wasthen isolated via vacuum. Flat, uniform, non-wetting surfaces aregenerated by pouring a PFPE-dimethacrylate (PFPE-DMA) containing2,2′-diethoxy-acetophenone over a silicon wafer and then subjected to UVlight (λ=365 nm) for 15 minutes while under a nitrogen purge. Followingthis, 50 μL of the PEG diacrylate solution is then placed on the nonwetting surface and the patterned PFPE mold placed on top of it. Thesubstrate is then placed in a molding apparatus and a small pressure isapplied to push out excess PEG-diacrylate solution. The entire apparatusis then subjected to UV light (J=365 nm) for 15 minutes while under anitrogen purge. Particles are observed after separation of the PFPEmold. The particles were harvested utilizing a sacrificial adhesivelayer and verified via DIC microscopy. These particles containing theCDI linker group were subsequently treated with and aqueous solution offluorescently tagged avidin. These particles were allowed to stir atroom temperature for four hours. These particles were then isolated viacentrifugation and rinsed with deionized water. These avidin labeledparticles were then treated with biotinylated-SUP-B8 (peptide specificto the specific surface immunoglobulin (sIg) known as the idiotype,which is distinct from the sIg of all of the patient's non-neoplasticcells) (see FIG. 55).

12.8 Controlled Mesh Density: Phantom Study and Cellular Uptake/MTTAssay

A patterned perfluoropolyether (PFPE) mold is generated by pouring aPFPE-dimethacrylate (PFPE-DMA) containing 2,2′-diethoxy-acetophenoneover a silicon substrate patterned with 3×3×11 um pillar shapes. Theapparatus is then subjected to UV light (λ=365 nm) for 15 minutes whileunder a nitrogen purge. The fully cured PFPE-DMA mold is then releasedfrom the silicon master. Separately, a poly(ethylene glycol) (PEG)diacrylate (n=9) is blended with 1 wt % of a photoinitiator,2,2′-diethoxy-acetophenone. 56 μL of PEG diacrylate monomer, 19 uL ofPEG monomethacrylate, 10 ug 2-acryloxyethyltrimethylammonium chloride(AETMAC), and 23 uL of a doxorubicin (26 mg/mL) are mixed. Flat,uniform, non-wetting surfaces are generated by pouring aPFPE-dimethacrylate (PFPE-DMA) containing 2,2′-diethoxy-acetophenoneover a silicon wafer and then subjected to UV light (λ=365 nm) for 15minutes while under a nitrogen purge. Following this, 50 μL of the PEGdiacrylate solution is then placed on the non-wetting surface and thepatterned PFPE mold placed on top of it. The substrate is then placed ina molding apparatus and a small pressure is applied to push out excessPEG-diacrylate solution. The entire apparatus is then subjected to UVlight (λ=365 nm) for 15 minutes while under a nitrogen purge. Particlesare observed after separation of the PFPE mold. The particles wereharvested utilizing a sacrificial adhesive layer and verified via DICmicroscopy. These particles were then dispersed in an aqueous solutionand exposed to NIH 3T3 mouse embryo fibroblasts cell lines at aconcentration of nanoparticles of 50 ug/mL. The particles and cells wereincubated for 48 hrs at 5% CO₂ at 37° C. The cells were thencharacterized via confocal and MTT assay.

12.9 Fabrication of Particles by Dipping Methods

A mold (5104) of size 0.5×3 cm with 3×3×8 micron patterned recesses(5106) was dipped into the vial (5102) with 98% PEG-diacrylate and 2%photo initiator solution. After 30 seconds the mold was withdrawn at arate of approximately 1 mm per second. The process is schematicallyshown in FIG. 56. Next, the mold was put into a UV oven, purged withnitrogen for 15 minutes and then cured for 15 minutes. The particleswere then harvested on a glass slide using cyanoacrylate adhesive. Noscum was detected and monodispersity of the particles was confirmedusing optical microscope, as shown in the image of FIG. 57. Furthermore,as evident in FIG. 57, the material contained in the recesses formed ameniscus with the sides of the recesses, as shown by reference number5402. This meniscus, when cured formed a lens on a portion of theparticle.

12.10 Fabrication of Particles by Droplet Moving

A mold (5200), 6 inch in diameter with 5×5×10 micron pattern recesses(5206) was placed on an incline surface having an angle of 20 degrees(5210) to the horizon. Next, a set of 100 micro liter drops (5204) wereplaced on the surface of the mold at a higher end. Each drop slid downthe mold leaving a trace of filled recesses (5208). The process isschematically shown in FIG. 58.

After all the drops reached the lower end of the mold, the mold was putin a UV oven, purged with nitrogen for 15 minutes and then cured for 15minutes. The particles were harvested on a glass slide usingcyanoacrylate adhesive. No scum was detected and monodispersity of theparticles was confirmed first using optical microscope (FIG. 59) andthen by scanning electron microscope (FIG. 59). Furthermore, as evidentin FIG. 59, the material contained in the recesses formed a meniscuswith the sides of the recesses, as shown by reference number 5502. Thismeniscus, when cured formed a lens on a portion of the particle.

Example 13 Control Mouse Studies

A patterned perfluoropolyether (PFPE) mold is generated by pouring aPFPE-dimethacrylate (PFPE-DMA) containing 2,2′-diethoxy-acetophenoneover a silicon substrate patterned with 200 nm shapes. The apparatus isthen subjected to UV light (λ=365 nm) for 15 minutes while under anitrogen purge. The fully cured PFPE-DMA mold is then released from thesilicon master. Separately, a poly(ethylene glycol) (PEG) diacrylate(n=9) is blended with 1 wt % of a photoinitiator,2,2′-diethoxy-acetophenone. 70 μL of PEG diacrylate monomer and 30 uL ofCDI-PEG monomer were mixed. Specifically, the CDI-PEG monomer wassynthesized by adding 1,1′-carbonyl diimidazole (CDI) to a solution ofPEG (n=400) monomethylacrylate in chloroform. This solution was allowedto stir overnight. This solution was then further purified by anextraction with cold water. The resulting CDI-PEG monomethacrylate wasthen isolated via vacuum. Flat, uniform, non-wetting surfaces aregenerated by pouring a PFPE-dimethacrylate (PFPE-DMA) containing2,2′-diethoxy-acetophenone over a silicon wafer and then subjected to UVlight (λ=365 nm) for 15 minutes while under a nitrogen purge. Followingthis, 50 μL of the PEG diacrylate solution is then placed on the nonwetting surface and the patterned PFPE mold placed on top of it. Thesubstrate is then placed in a molding apparatus and a small pressure isapplied to push out excess PEG-diacrylate solution. The entire apparatusis then subjected to UV light (λ=365 nm) for 15 minutes while under anitrogen purge. Particles are observed after separation of the PFPEmold. The particles were harvested utilizing a sacrificial adhesivelayer and verified via DIC microscopy. These particles containing theCDI linker group were subsequently treated with and aqueous solution offluorescently tagged avidin. These particles were allowed to stir atroom temperature for four hours. These particles were then isolated viacentrifugation and rinsed with deionized water. These avidin labeledparticles were then treated with biotin. A solution (2.5 mgavidin/biotin nanoparticles/200 uL saline) was administered to 4 Neutransgenic mice (2.5 mg avidin/biotin nanoparticles/200 uL saline) every14 days for 2 cycles (total 28 days) versus a control group 4 Neutransgenic mice that was treated with 200 uL saline every 14 days for 2cycles (total 28 days). Both sets of mice seemed to produce no adverseside effects from either treatment.

Example 14 Particle Fabrication

14.1 Synthesis of 200 nm Cationic PEG Particles for Pharmacokinetics

A patterned perfluoropolyether (PFPE) mold is generated by pouring aPFPE-dimethacrylate (PFPE-DMA) containing 2,2′-diethoxy-acetophenoneover a silicon substrate patterned with 200 nm shapes. The apparatus ispurged with nitrogen for 10 minutes, and then subjected to UV lightQ=365 nm) for 6 minutes while under a nitrogen purge. The fully curedPFPE-DMA mold is then released from the silicon master, and blown withair to remove dust. Separately, a solution containing 84 mol % PEGdiacrylate, 5 mol % PEG monoacrylate, 10 mol % aminoethylmethacrylatehydrochloride, and 1 mol % photoinitiator was prepared. The mold wasplaced in a fume hood and the hydrogel-monomer solution was atomizedonto mold. A polyethylene sheet was then placed over the mold andbubbles were removed by manual pressure with a roller. The polyethylenecover was slowly removed to fill the particle chambers. Themold/solution combination was placed into a UV curing chamber, purgedfor 10 minutes with nitrogen, and UV cured for 0.8 minutes. Theparticle/mold combination was placed in the spin coater and the spincoater started at approx 1000 rpm. Approx 20 mls of nitro-cellulose wasput into the center of the spinning mold and left to cure for 1 minutewhile rotating. The nitro-cellulose is then carefully lifted off themold with particles attached and placed in a vial. Acetone is then addedto dissolve the cellulose and leave the particles. The particles werepurified via centrifugation, and then strained through a 100 meshscreen. The remaining acetone is carefully aspirated and the particlesdried under nitrogen.

14.2 Synthesis of 200 nm Triacrylate Particles

Molds suitable for PRINT fabrication of 200×200×200 nm particles wereprepared by pooling end-functionalized PFPE dimethacrylate precursorcontaining 0.1% diethoxyacetophenone (DEAP) photoinitiator onto a mastertemplate containing 200×200×200 nm posts. The telechelic PFPE precursorwas UV polymerized under a blanket of nitrogen into a cross-linkedrubber (the “mold”). The mold was then peeled away from the master,revealing 200×200×200 nm patterned cavities in the mold. 1 parttrimethylolpropane triacrylate containing 10% DEAP (“triacrylate resin”)was then dissolved in 10 parts methanol and spray-coated onto thepatterned side of the mold until full coverage was achieved. A thinpolyethylene sheet was placed over the patterned side of the mold andsealed to the mold by manually applying a small amount of pressure. Thepolyethylene sheet was then slowly peeled away from the mold (˜1mm/sec), allowing capillary filling of the cavities in the mold. Excesstriacrylate resin was gathered at the PFPE/polyethylene interface andremoved from the mold as the polyethylene sheet was peeled away. Oncethe polyethylene sheet was fully peeled away from the mold, any residualmacroscopic droplets of triacrylate resin were removed from the mold.The triacrylate resin filling the patterned cavities in the mold wasthen UV polymerized under a blanket of nitrogen for about 5 minutes.Collodion solution (Fisher Scientific) was then spin-cast onto thepatterned side of the mold to produce a robust nitrocellulose-basedfilm. This film was then peeled away from the mold to remove particlesby adhesive transfer to the nitrocellulose film. The nitrocellulose filmwas then dissolved in acetone. The particles were purified from thedissolved nitrocellulose by a repetitive process of sedimenting theparticles, decanting nitrocellulose/acetone solution, and resuspensionof the particles in clean acetone. This process was repeated until allthe nitrocellulose was separated from the particles.

It will be understood that various details of the presently disclosedsubject matter can be changed without departing from the scope of thepresently disclosed subject matter. Furthermore, the foregoingdescription is for the purpose of illustration only, and not for thepurpose of limitation.

What is claimed is:
 1. A synthetic particle, comprising: a substantiallydisc shaped, molded synthetic polymer particle having largest lineardimension of between about 5 μm to about 10 μm and a modulus of lessthan about 1 MPa, wherein the particle can pass through an opening ofless than about 3 μm.
 2. The synthetic particle of claim 1, wherein thesynthetic polymer comprises a biocompatible polymer.
 3. The syntheticparticle of claim 1, wherein the synthetic polymer comprisespoly(ethylene glycol), poly(lactic acid), poly(lactide-co-glycolide),poly(ethylene glycol) diacrylate, or trimethylolpropane triacrylate. 4.The synthetic particle of claim 1, wherein the particle furthercomprises a cargo selected from the group consisting of a therapeuticagent, a biologic, a diagnostic agent, hemoglobin, and an imaging agent.5. The synthetic particle of claim 4, wherein the cargo is capable ofbinding and releasing oxygen.
 6. The synthetic particle of claim 1,wherein the opening is an orifice, an opening of a tube or a lumen.
 7. Adrug delivery particle, comprising: a substantially disc shaped, moldedsynthetic polymer particle having a largest linear dimension less thanabout 10 μm and a modulus of less than about 1 MPa, wherein the particlecan pass through an opening less than about 60% the largest lineardimension of the particle.
 8. The drug delivery particle of claim 7,wherein the particle can pass through an opening of less than about37.5% the largest linear dimension of the particle.
 9. The drug deliveryparticle of claim 7, wherein the particle can pass through an opening ofless than about 30% the largest linear dimension of the particle. 10.The drug delivery particle of claim 7, wherein the synthetic polymercomprises a biocompatible polymer.
 11. The drug delivery particle ofclaim 7, wherein the synthetic polymer comprises poly(ethylene glycol),poly(lactic acid), poly(lactide-co-glycolide), poly(ethylene glycol)diacrylate, or trimethylolpropane triacrylate.
 12. The drug deliveryparticle of claim 7, wherein the particle further comprises a cargoselected from the group consisting of a therapeutic agent, a biologic, adiagnostic agent, hemoglobin, and an imaging agent.
 13. The drugdelivery particle of claim 7, wherein the opening is an orifice, anopening of a tube or a lumen.